CN104560870A - Method for preparing decidua mesenchymal stem cell - Google Patents
Method for preparing decidua mesenchymal stem cell Download PDFInfo
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- CN104560870A CN104560870A CN201410795200.0A CN201410795200A CN104560870A CN 104560870 A CN104560870 A CN 104560870A CN 201410795200 A CN201410795200 A CN 201410795200A CN 104560870 A CN104560870 A CN 104560870A
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Abstract
The invention provides a method for preparing a decidua mesenchymal stem cell. The method comprises the following steps: performing placenta asepsis by taking the placenta of a male full-term fetus as a raw material; separating a decidual tissue; identifying the decidual tissue and identifying whether the decidua mesenchymal stem cell comes from a maternal tissue; culturing the decidua mesenchymal stem cell; freeze-storing the decidua mesenchymal stem cell; and reviving the decidua mesenchymal stem cell. The method provided by the invention has the technical characteristics that the sampled fetus is male fetus placenta; after the decidual tissue is obtained, sex determination is performed to identify whether the decidual tissue comes from a parent body and is free of pollution of a daughter tissue; by adopting a bacterial pollution prevention method, the pollution possibility is reduced from a sampling source; the surface is washed for a plurality of times, so that the pollution possibility is effectively reduced; a single enzyme is used for digestion to simplify the procedures; a serum-free medium is used for culture to reduce the use of an animal source component; the cell is stable in performance; a long-term in-vitro culture process of the decidua mesenchymal stem cell can be maintained; and the cellular morphology, multiplication capacity, MSC surface marking expression capacity, differentiative capacity and the like of the cell can be maintained.
Description
Technical field
The present invention relates to a kind of method preparing decidua mescenchymal stem cell, belong to stem cells technology field.
Background technology
Mescenchymal stem cell (mesenchymal stem cell, MSC) be derive to grow a mesoblastic class multipotential stem cell, there is self and multi-lineage potential, it can be divided into polytype histocyte under specific inductive condition, can form the Various Tissues such as bone, cartilage, fat, cardiac muscle.Mescenchymal stem cell is mainly derived from marrow the earliest, but mescenchymal stem cell (BMMSC) content in people's marrow is extremely low.In recent years, the existing mesenchymal cell possessing ancestral cells feature is separated in the Various Tissues such as peripheral blood, dental pulp, cartilage, muscle, umbilical cord and placenta.The umbilical cord mesenchymal stem cells, amnion mesenchymal stem cell and the chorion mescenchymal stem cell that come from daughter can be separated from placenta tissue, and derive from the decidua mescenchymal stem cell of parent.The mesenchymal cell of adult can be divided into the multiple cell with specific function under different conditions, as scleroblast, chondrocyte, adipocyte, myocardial cell, myocyte etc., and, there is huge potential medical treatment and store value.The umbilical cord of placenta, amnion and chorion can extract mescenchymal stem cell, as the transplanting or frozen of child's own cells.And the mescenchymal stem cell that the decidua of placenta extracts, derive from parent, may be used for the transplanting or frozen of mother's own cells, pointed.
Decidua mescenchymal stem cell often adopts enzymic digestion decidua tissue to obtain, the multiplex culture medium culturing containing 10%FBS, and the multiplex frozen storing liquid containing 10%DMSO is frozen.
In prior art, the method preparing decidua mescenchymal stem cell can have the following disadvantages usually:
1) prior art of decidua tissue separation, cannot peel off the simple decidua tissue coming from parent, total mixed placental chorion tissue or amnion tissue deriving from daughter.
2) prior art that placenta collection and decidua are separated lacks effective decreasing pollution measure, anti-pollution measure limited use, original cuiture pollution rate is high, lack the method for decreasing pollution from gathering source, at present adding microbiotic in cultivation link can only suppress contaminated bacteria to be bred in culturing process, can not effective decreasing pollution;
2) existing enzymic digestion method is complicated, needs repeatedly enzymic digestion;
3) operation such as enzymic digestion, cryopreservation resuscitation likely causes cell injury to cell;
4) in stem cell culturing process, cell is easily old and feeble, cell is flat, increment is slow or stop propagation, lose differentiation capability etc., not yet there are method or technical experience to set forth at present and effectively maintain decidua mescenchymal stem cell form, prevent cell senescence, maintain cell and retain differentiation capability in breeding;
5) Telomerase inactivation may be there is after mescenchymal stem cell Long Term Passages, oncogene expression increase expression of tumor suppressor gene decline, karyotypic alteration equivalent risk, lack the research data of decidua mescenchymal stem cell this respect at present.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of method preparing simple decidua mescenchymal stem cell is provided.
The method preparing decidua mescenchymal stem cell of the present invention, comprises the following steps:
1) use male sex's term fetus placenta of giving a birth in discharge 0 ~ 30min as raw material; Rinse placenta appearance 1-3 time with placenta scavenging solution, placenta is soaked in placenta scavenging solution;
2) in 48h, the placenta be immersed in placenta scavenging solution is transported to laboratory, takes out placenta, again with scavenging solution cleaning 1-3 time, again from careful separation parietal decidua bottom placenta, after cleaning up, put into scavenging solution and soak 0 ~ 30min, then decidua tissue is shredded into 1 ~ 3mm
3the tissue block of size;
3) decidua tissue is kept sample carry out sex identification, determine that the decidua tissue gathered comes from parent merely;
4) decidua mescenchymal stem cell is obtained by collagenase digestion from decidua tissue block;
5) serum free medium is used to cultivate decidua mescenchymal stem cell, wherein P0 covers with to 80% density for decidua mescenchymal stem cell, use 0.25% trysinization, digestion time is 3-5min, in P0 generation, afterwards, cell goes down to posterity in 48-72h after operation of going down to posterity, and cell density 80%-90% when requiring to go down to posterity, after going down to posterity, cell is by (3-8) × 10
3individual/cm
2density is inoculated, and notes observing the change of substratum pH value in culturing process, adds fresh culture once turn yellow and removes the old substratum of culture vessel moderate, and cell went down to posterity from P0 generation, reached any generation in the middle of P1 ~ P30;
Wherein,
Described step 1) and step 2) in scavenging solution be 0.9% physiological saline;
Described step 4) in collagenase digestion in the collagenase II of collagenase used to be final concentration be 1-5mg/ml;
Described step 5) in serum free medium consist of the following composition: DMEM/F12, PDGF-BB, bFGF, (TGF)-β 1, EGF and Transferrins,iron complexes Transferrin; Wherein, PDGF-BB content is 50 ~ 100ng/mL; BFGF content is 0 ~ 50ng/mL; (TGF)-β 1 content is 0 ~ 20ng/mL; EGF content is 0 ~ 30ng/mL; Transferrins,iron complexes Transferrin content is 2.0 ~ 4.5mg/mL.
Preferably,
Described step 1) and step 2) in scavenging solution also containing microbiotic, said microbiotic is penicillin, Streptomycin sulphate or amphotericin B.
Described step 4) in the step of collagenase digestion as follows: decidua tissue block is put into 37 DEG C of temperature, vibration digestion 15 ~ 60min, add the physiological saline that volume is tissue block volume 1 ~ 5 times, with the centrifugal 5min of the rotating speed of 2500 turns/min, remove supernatant, bottom is digested after product, proceed to culture vessel, the ratio adding 10ml serum free medium in 1ml decidua tissue block adds serum free medium, described step 4) in serum free medium consist of the following composition: DMEM/F12, PDGF-BB, bFGF, (TGF)-β 1, EGF and Transferrins,iron complexes Transferrin, wherein, PDGF-BB content is 50 ~ 100ng/mL, bFGF content is 0 ~ 50ng/mL, (TGF)-β 1 content is 0 ~ 20ng/mL, EGF content is 0 ~ 30ng/mL, Transferrins,iron complexes Transferrin content is 2.0 ~ 4.5mg/mL.
Described step 4) in serum free medium consist of the following composition: DMEM/F12+80ng/mLPDGF-BB+20ng/mL bFGF+10ng/mL (TGF)-β 1+10ng/mL EGF+3.0mg/mL Transferrins,iron complexes.
Described step 4) in serum free medium in be also added with microbiotic, said microbiotic is penicillin, Streptomycin sulphate or amphotericin B.
Described step 5) in serum free medium consist of the following composition: DMEM/F12+80ng/mLPDGF-BB+20ng/mL bFGF+10ng/mL (TGF)-β 1+10ng/mL EGF+3.0mg/mL Transferrins,iron complexes.
Described step 5) in P0 be also added with microbiotic in the serum free medium of cell cultures, said microbiotic is penicillin, Streptomycin sulphate or amphotericin B.
The cryopreservation methods of described decidua mescenchymal stem cell is as follows: every 10
6-10
7cell adds 1ml frozen storing liquid, puts into freezing storing box, proceeds to-80 DEG C of refrigerators, proceeds in-196 DEG C of liquid nitrogen or gas nitrogen and save backup after 12 ~ 24h; Described frozen storing liquid is made up of basal liquid and perviousness cryoprotectant, and said perviousness cryoprotection agent concentration is 1-1.4mol/L, and said basal liquid is nutrient solution DMEM/F12; Said perviousness cryoprotectant is dimethyl sulfoxide (DMSO).
The method preparing decidua mescenchymal stem cell of the present invention, has following technique effect:
1) selection fetus is the mature placenta of the male sex is raw material, can take precautions against pollution method, from source decreasing pollution probability, repeatedly rinse surface, effective decreasing pollution probability;
2) limit decidua to draw materials region: bottom placenta, softly strip most surface decidua tissue, reduce and mix cell contamination; Sex identification is carried out to the decidua tissue stripped down simultaneously, guarantee that the decidua tissue stripped down does not have the pollution of its hetero-organization of placenta.
3) in this patent method be separated decidua mescenchymal stem cell time, adopt single enzyme digestion, can flow process be simplified;
4) use serum free medium to cultivate and reduce the use of animal source composition, cell according to said method can be passaged to P30 from P0 generation and maintain stable performance, decidua mescenchymal stem cell long-term cultivation process in vitro can be maintained, maintain cellular form, multiplication capacity, the expression of MSC surface markers, differentiation capability, also can maintain decidua mescenchymal stem cell long-term cultivation process in vitro, maintenance Telomerase Expression, oncogene stably express, caryogram are stablized;
5) in present method enzymic digestion and cryopreservation resuscitation to cell fanout free region.
Accompanying drawing explanation
Fig. 1 is the aspect graph of obtained decidua mescenchymal stem cell;
Fig. 2-1 is the sex identification result of decidua tissue and other placenta tissues be separated;
Fig. 2-2 is surface markers detected 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 detected result figure through the postdigestive decidua mescenchymal stem cell of digestive ferment;
Fig. 5 adopts the P0 ~ P10 of serum free medium cultivation for decidua mescenchymal stem cell aspect graph;
Fig. 6 be adopt serum free medium to cultivate P2, P5 and P10 for decidua mescenchymal stem cell, with aged cells and undifferentiated cell form contrast figure;
Fig. 7 is that growth curve measures detected result figure;
Fig. 8 is skeletonization, becomes fat, becomes chondrocyte induction differentiation detected result figure;
Fig. 9 is flow cytometer detection result figure;
Figure 10 is Telomerase activity result figure;
Figure 11 is oncogene detected result figure;
Figure 12 is G Banded karyotype analytical results figure.
Embodiment
Reagent used in embodiment is purchased from the manufacturer in bracket after reagent name.
Embodiment 1 prepares decidua mescenchymal stem cell
The method preparing decidua mescenchymal stem cell in the present embodiment, comprises the following steps:
1) use male sex's term fetus placenta of giving a birth in discharge 0 ~ 30min as raw material; Operator sterile gloves taking placenta, prevent placenta from contacting the object (as ground etc.) that may bring pollution in operating process, appearance is rinsed 3 times with placenta scavenging solution, scavenging solution is 0.9% physiological saline (world, Guizhou), can add in scavenging solution or not add microbiotic, the microbiotic added is penicillin (gibco), Streptomycin sulphate (gibco), amphotericin B (sigma).Use sterile gauze wipe surfaces, remove surperficial bloodstain, be soaked in the absence of liquid drippage of placenta surface in the placenta collecting cassette containing scavenging solution, in 48h, the placenta be immersed in scavenging solution be transported to laboratory.
2) after placenta is transported to laboratory, in the middle of the plate being positioned over 40cm × 40cm × 20cm (long × wide × high), appearance is rinsed 3 times with aseptic scavenging solution, scavenging solution is 0.9% physiological saline (world, Guizhou), can add in scavenging solution or not add microbiotic, the microbiotic added is penicillin (gibco), Streptomycin sulphate (gibco), amphotericin B (sigma).Be inverted placenta, bottom placenta, softly strip decidua outermost layer tissue, the decidua tissue of clip is cleaned with scavenging solution, and scavenging solution is 0.9% physiological saline (world, Guizhou), adds scavenging solution and soaks 30min.Decidua tissue is shredded into 1 ~ 3mm
3the tissue block of size.
After above-mentioned placenta source starts process, in follow-up culturing process, pollution rate effectively reduces.Do not sterilize in placenta source, subsequent contamination incidence is (25.3 ± 7.2%), carries out pollution incidence be reduced to (2.4 ± 1.5%) by present method, significantly reduces to pollute to occur.
3) decidua tissue is kept sample carry out sex identification, determine that the decidua tissue gathered comes from parent merely
Sex appraisal method: get need qualification tissue or cell according to test kit (solarbio, D1800) method extracts total genome, add the conserved sequence SYB on the reagent expansion Y chromosome of PCR kit, agarose (takara) gel electrophoresis is carried out to result.As can be seen from the results, decidua tissue SYB is negative, comes from maternal tissue and decidua, and other positives be all autologous tissue.
Qualification result is as shown in Fig. 2-1, and the conserved sequence that the decidua tissue deriving from parent shows as on Y chromosome is feminine gender, and is positive from the tissue characterization result of chorion, amnion, umbilical cord.Can find out, decidua tissue derives from parent.
4) decidua mescenchymal stem cell is separated from decidua tissue block
The volume ratio adding 1-5ml digestive ferment by every 1ml tissue block adds digestive ferment (gibco), the collagenase II (gibco) of said digestive ferment to be final concentration be 1-5mg/ml.
Digestion step is as follows: the decidua tissue block having added digestive ferment is placed in 37 DEG C of temperature, and vibration digestion 15-60min, vibrating is provided by vibrator.Adding volume is again tissue block volume 1-5 0.9% physiological saline (world, Guizhou) doubly, and 2500 leave heart 5min, remove supernatant.Bottom is digested after product, proceeds to culture vessel, the ratio adding 10ml serum free medium (gibco) in 1ml decidua tissue block adds serum free medium.Rear continuation cultivation 5 ~ 20 days are climbed out of to decidua mescenchymal stem cell.
The composition of serum free medium is: DMEM/F12 (invitrogen)+80ng/mLPDGF-BB (gibco)+20ng/mLbFGF (invitrogen)+10ng/mL (TGF)-β 1 (Peprotech)+10ng/mL EGF (gibco)+3.0mg/mL Transferrins,iron complexes (sigma).
Can add containing microbiotic in serum free medium, microbiotic is penicillin (gibco), Streptomycin sulphate (gibco), amphotericin B (sigma).
Adopt aforesaid method, save the operating time of every part of tissue, reduce the consumable reagent such as centrifuge tube, physiological saline and use; Digestion time shortens, and shortens to 60min by the 90min of two-step approach.
5) decidua mescenchymal stem cell is cultivated
Decidua mescenchymal stem cell serum free medium is cultivated.
The composition of serum free medium is: DMEM/F12 (invitrogen)+80ng/mLPDGF-BB (gibco)+20ng/mLbFGF (invitrogen)+10ng/mL (TGF)-β 1 (Peprotech)+10ng/mL EGF (gibco)+3.0mg/mL Transferrins,iron complexes (sigma).
The serum free medium that cultivation P0 uses for decidua mescenchymal stem cell can add microbiotic, and microbiotic is penicillin (gibco), Streptomycin sulphate (gibco), amphotericin B (sigma).
Use serum free medium to cultivate decidua mescenchymal stem cell, P0 covers with to 80% density for decidua mescenchymal stem cell, and use 0.25% pancreatin (gibco) to digest, digestion time is 3-5min.Generation after P0 generation uses not containing antibiotic culture medium culturing.
In P0 generation, cell went down to posterity in 48 ~ 72h after operation of going down to posterity afterwards, and when requiring to go down to posterity, cell density reaches 80%-90%, and after going down to posterity, cell is by 3 × 10
3-8 × 10
3individual/cm2 density inoculation.Note in culturing process observing the change of substratum pH value, once PH turns yellow add fresh culture.Every 24h takes pictures observed and recorded cellular form.
As shown in Figure 1, can find out that to obtain passage form good;
As shown in Fig. 2-2, surface markers meets ISCT to mescenchymal stem cell examination criteria;
As shown in Figure 3, after digestive ferment direct effect cell, cell maintains good form;
As shown in Figure 4, after digestion, Cell growth ability is uninfluenced;
As shown in table 1, surface markers is uninfluenced.Illustrate that this Digestive system formula does not have an impact to mescenchymal stem cell in decidua and decidua mescenchymal stem cell.
Table 1
As shown in Figure 5, the P0 ~ P10 adopting serum free medium to cultivate is good for decidua mescenchymal stem cell form.
P0 ~ P30 generation centre is any once all can carry out morphologic observation, propagation test, differentiation-inducing, streaming, Telomerase, oncogene expression, caryogram detection.
1. morphologic observation record cellular form, contrasts with aged cells and undifferentiated cell form, and detected result as shown in Figure 6, can find out that after going down to posterity, cellular form remains good, unaged;
2. breed test to comprise: growth curve measures, detected result as shown in Figure 7, can find out that after repeatedly going down to posterity, cell maintains stronger proliferation activity;
3. differentiation-inducing detection comprises: skeletonization, one-tenth fat, one-tenth cartilage detect, and detected result as shown in Figure 8, can find out that after repeatedly going down to posterity, cell maintains differentiation capability;
4. flow cytometer detection comprises: CD73, CD90, CD105, CD14, CD34, CD45, CD79-α, HLA-DR, and detected result as shown in Figure 9, can find out that after repeatedly going down to posterity, cell surface marker expression remains stable, meets MSC examination criteria;
5. Telomerase activity method is: adopt
telomerase DetectionKit (millipore) test kit detects, and detected result as shown in Figure 10, can find out that after repeatedly going down to posterity, cell does not lose telomerase activation;
6. oncogene detects and comprises: Real-time PCR detects oncogene (c-Myc, c-fos, k-ras) and cancer suppressor gene (P53, P21, RB, P16) express, detected result as shown in figure 11, can find out that oncogene and expression of tumor suppressor gene are expressed more stable;
7. caryogram detects: adopt the analysis of G Banded karyotype, and as shown in figure 12, can find out that cell caryogram after repeatedly going down to posterity is normal, be both the caryogram that cell caryogram should be women, sex chromosome is XX to detected result.
Therefore, the decidua mescenchymal stem cell cell that preparation method of the present invention obtains is in vitro in succeeding generations, cellular form, multiplication capacity, the expression of MSC surface markers, differentiation capability can be maintained, also can maintain decidua mescenchymal stem cell long-term cultivation process in vitro, maintenance Telomerase Expression, oncogene stably express, caryogram are stablized.
6) frozen decidua mescenchymal stem cell:
Every 10
6~ 10
7cell adds 1ml frozen storing liquid, puts into freezing storing box, proceeds to-80 DEG C of refrigerators, proceeds in-196 DEG C of liquid nitrogen or gas nitrogen and preserve after 12-24h.
Decidua mesenchymal stem cell cryopreserving liquid is made up of basal liquid and perviousness cryoprotectant, and said perviousness cryoprotection agent concentration is 1mol/L, and said basal liquid is nutrient solution DMEM/F12 (invitrogen); Said perviousness cryoprotectant is dimethyl sulfoxide (DMSO) (gibco).
7) recovery decidua mescenchymal stem cell:
The decidua mescenchymal stem cell that will recover or decidua tissue block take out from liquid nitrogen container, put into 37 DEG C of water-baths and hatch 2-3min, after the washing of 0.9% physiological saline (world, Guizhou) of 4 DEG C of precoolings, centrifugally remove supernatant, add serum free medium.
Serum-free culture based component used and step 5 in this step) in identical.
Cryopreservation resuscitation success ratio: cryopreservation resuscitation 111 times, success ratio 100%.Cellular form is good, flow cytometer detection is qualified, and it is qualified that differentiation capability detects.
The concrete grammar step of the various detections related in the present invention is as follows respectively.Involved parameter is by following Instrument measuring: OLYMPUS inverted microscope, Lycra just put (laica) microscope, FACSAira flow cytometer, ultraviolet-visible pectrophotometer, Biorad fluorescent PCR instrument, BioradPCR instrument, incubator, cryogenic refrigerator, liquid nitrogen container etc.
(1) OLYMPUS microscope is taken pictures
The P2 turned out merges to more than 80-90% for decidua mescenchymal stem cell, and take pictures under being placed on inverted microscope, cell shape is short fusiformis, and size is homogeneous, marshalling, as shown in Figure 6.
(2) drafting of growth curve
Get the cell prepared, digest to single cell suspension; Serum free medium adjustment concentration to 1 × 10
4/ ml, point adds in 96 orifice plates; If 13 groups are often organized 8 multiple holes, every hole 200 μ l, is placed in 37 DEG C, cultivates containing the CO2 of 5%, the incubator of saturated humidity; Within every 2 days, change liquid, respectively after being cultured to 1-13 days, the concentration that every hole adds 20 μ l is the tetrazolium bromide (MTT) (sigma, M5655) of 5mg/mL, continues to cultivate; Carefully culture supernatant is removed after 4 hours; Every hole adds the dimethyl sulfoxide (DMSO) (takara, 67-68-5) of 100 μ l, and micro oscillator shakes 5 minutes; Put the absorbance value under the upper survey of microplate reader (Thermo) 570nm or 490nm, after statistical analysis, draw out growth curve.After digestion, cell growth curve as shown in Figure 4, and after repeatedly going down to posterity, cell growth curve as shown in Figure 7.
(3) Multidirectional Differentiation ability qualification
By 1 × 10
4the P2 of/ml for cell suspension inoculation in 24 well culture plates, 0.5ml/ hole; Cytogamy, to 60-80%, changes osteogenic induction perfect medium (gibco) into, 0.5ml/ hole; Within every 3-4 days, full dose changes induction broth; Induce after 24 days, take pictures after sodium alizarinsulfonate (traditional Chinese medicines group) dyeing, have a large amount of calcium tubercle to produce as seen, as shown in Figure 8.
By 1 × 10
4the P2 of/m for cell suspension inoculation in 24 well culture plates, 0.5ml/ hole; Cytogamy, to 80-90%, changes adipogenic induction perfect medium (gibco) into, 0.5ml/ hole; Within every 3-4 days, full dose changes induction broth; Induce after 14 days, take pictures after oil red O (gibco) dyeing, all have fat to drip generation in visible all cells, as shown in Figure 8.
By 5 × 10
5p2 for cell suspension inoculation in the centrifuge tube of 15ml, centrifugal rear cultivation, changes into chondrocyte induction perfect medium (gibco) for second day into, 0.5ml/ hole; Within every 3-4 days, full dose changes induction broth; Induce after 21 days, section, alcian blue (traditional Chinese medicines group) dyeing is taken pictures, and all has cartilage to produce, as shown in Figure 8 in visible all cells.
(4) FCM analysis
By 5 × 10
6cell dissociation becomes single cell suspension; With PBS washing twice rear adjustment concentration to 1 × 105/ml, flow cytometer (BD, FACSAira) cell surface marker positive indication CD90, CD73 and CD105 is detected, negative indication CD34, CD14, CD45, CD79a, HLA-DR, positive cell rate is all more than 95%, negative indication meets lower than 2%, meets the feature of mescenchymal stem cell, as shown in Figure 9.
(5) Telomerase activity
Get 1*10
6cell according to
the specification sheets of Telomerase Detection Kit (milipore) test kit extracts sample and identifies, detected result as shown in Figure 10, can find out that after repeatedly going down to posterity, cell does not lose telomerase activation.
(6) oncogene and tumor repressive gene detects
Get decidua mescenchymal stem cell 1x10
6cell extraction RNA (extracting test kit specification sheets with reference to QIAGEN RNA), reverse transcription is CDNA (TOYOBO First Strand cDNA Synthesis Kit), adds oncogene and cancer suppressor gene and carries out quantitative fluorescent PCR (biorad) and detect.Use 2
-△ △ Ctmethods analyst experimental data, whether the expression detecting relevant proto-oncogene and cancer suppressor gene there is noticeable change.Real-time PCR detects oncogene (c-Myc, c-fos, k-ras) and cancer suppressor gene (P53, P21, RB, P16) is expressed, and detected result as shown in figure 11, can find out that oncogene and expression of tumor suppressor gene are expressed more stable.
(7) karyotyping
Get the decidua mescenchymal stem cell colchicine process in cultivation, digestion, collected by centrifugation, PBS washs.Hypotonic KCL (0.075M) processes, treatment time is at 20-40 minute, drip a small amount of stationary liquid, centrifugal 1500rpm/5min removes supernatant, retain 1ml supernatant, piping and druming is hanged gently, slowly adds stationary liquid (methyl alcohol: Glacial acetic acid is according to the mixing of 3:1 ratio), limit edged vibration, until full packages.Centrifugally remove supernatant, pat gently and hang, slowly add stationary liquid, the vibration of limit edged, until full packages.4 spend night, centrifugally remove supernatant.Drip sheet, slide glass is placed on 0 degree of ice-water bath in advance, drips sheet after taking out slide from eminence.Overdo fast or dry air, after Giemsa staining, under mirror, (laica) observes.
Claims (8)
1. prepare a method for decidua mescenchymal stem cell, it is characterized in that, comprise the following steps:
1) use male sex's term fetus placenta of giving a birth in discharge 0 ~ 30min as raw material; Rinse placenta appearance 1-3 time with placenta scavenging solution, placenta is soaked in placenta scavenging solution;
2) in 48h, the placenta be immersed in placenta scavenging solution is transported to laboratory, takes out placenta, again with scavenging solution cleaning 1-3 time, again from careful separation parietal decidua bottom placenta, after cleaning up, put into scavenging solution and soak 0 ~ 30min, then decidua tissue is shredded into 1 ~ 3mm
3the tissue block of size;
3) decidua tissue is kept sample carry out sex identification, determine that the decidua tissue gathered comes from parent merely;
4) decidua mescenchymal stem cell is obtained by collagenase digestion from decidua tissue block;
5) serum free medium is used to cultivate decidua mescenchymal stem cell, wherein P0 covers with to 80% density for decidua mescenchymal stem cell, use 0.25% trysinization, digestion time is 3-5min, in P0 generation, afterwards, cell goes down to posterity in 48-72h after operation of going down to posterity, and cell density 80%-90% when requiring to go down to posterity, after going down to posterity, cell is by (3-8) × 10
3individual/cm
2density is inoculated, and notes observing the change of substratum pH value in culturing process, adds fresh culture once turn yellow and removes the old substratum of culture vessel moderate, and cell went down to posterity from P0 generation, reached any generation in the middle of P1 ~ P30;
Wherein,
Described step 1) and step 2) in scavenging solution be 0.9% physiological saline;
The collagenase II of collagenase used in the collagenase digestion in described step 4) to be final concentration be 1-5mg/ml;
Serum free medium in described step 5) consists of the following composition: DMEM/F12, PDGF-BB, bFGF, (TGF)-β 1, EGF and Transferrins,iron complexes Transferrin; Wherein, PDGF-BB content is 50 ~ 100 ng/mL; BFGF content is 0 ~ 50 ng/mL; (TGF)-β 1 content is 0 ~ 20ng/mL; EGF content is 0 ~ 30 ng/mL; Transferrins,iron complexes Transferrin content is 2.0 ~ 4.5 mg/mL.
2. the method preparing decidua mescenchymal stem cell according to claim 1, is characterized in that, described step 1) and step 2) in scavenging solution also containing microbiotic, said microbiotic is penicillin, Streptomycin sulphate or amphotericin B.
3. the method preparing decidua mescenchymal stem cell according to claim 1, it is characterized in that, the step of the collagenase digestion in described step 4) is as follows: decidua tissue block is put into 37 DEG C of temperature, vibration digestion 15 ~ 60min, add the physiological saline that volume is tissue block volume 1 ~ 5 times, with the centrifugal 5min of the rotating speed of 2500 turns/min, remove supernatant, bottom is digested after product, proceed to culture vessel, the ratio adding 10ml serum free medium in 1ml decidua tissue block adds serum free medium, serum free medium in described step 4) consists of the following composition: DMEM/F12, PDGF-BB, bFGF, (TGF)-β 1, EGF and Transferrins,iron complexes Transferrin, wherein, PDGF-BB content is 50 ~ 100 ng/mL, bFGF content is 0 ~ 50 ng/mL, (TGF)-β 1 content is 0 ~ 20ng/mL, EGF content is 0 ~ 30 ng/mL, Transferrins,iron complexes Transferrin content is 2.0 ~ 4.5 mg/mL.
4. the method preparing decidua mescenchymal stem cell according to claim 3, it is characterized in that, the 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,iron complexes.
5. the method preparing decidua mescenchymal stem cell according to claim 3 or 4, is characterized in that, is also added with microbiotic in the serum free medium in described step 4), and said microbiotic is penicillin, Streptomycin sulphate or amphotericin B.
6. the method preparing decidua mescenchymal stem cell according to claim 1, it is characterized in that, the 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,iron complexes.
7. the method preparing decidua mescenchymal stem cell according to claim 1 or 6, it is characterized in that, P0 in described step 5) is also added with microbiotic in the serum free medium of cell cultures, and said microbiotic is penicillin, Streptomycin sulphate or amphotericin B.
8. the method preparing decidua mescenchymal stem cell according to claim 1, is characterized in that, the cryopreservation methods of described decidua mescenchymal stem cell is as follows: every 10
6-10
7cell adds 1ml frozen storing liquid, put into freezing storing box, proceed to-80 DEG C of refrigerators, proceed to after 12 ~ 24h in-196 DEG C of liquid nitrogen or gas nitrogen and save backup, described frozen storing liquid is made up of basal liquid and perviousness cryoprotectant, said perviousness cryoprotection agent concentration is 1-1.4 mol/L, and said basal liquid is nutrient solution DMEM/F12; Said perviousness cryoprotectant is dimethyl sulfoxide (DMSO).
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| 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 |
| CN106957814A (en) * | 2017-05-19 | 2017-07-18 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of amnion mesenchymal stem cell culture medium and culture amnion mesenchymal stem cell |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| CN113136364A (en) * | 2021-04-13 | 2021-07-20 | 广东唯泰生物科技有限公司 | Preparation method and recovery method of decidua-muralis mesenchymal stem cells |
| 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 |
| WO2022217869A1 (en) * | 2021-04-13 | 2022-10-20 | 广东唯泰生物科技有限公司 | Pariduval mesenchymal stem cell culture medium for treating tumors, and co-culture method |
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| 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 |
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| 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 |
| 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 |
| CN113136364A (en) * | 2021-04-13 | 2021-07-20 | 广东唯泰生物科技有限公司 | Preparation method and recovery method of decidua-muralis mesenchymal stem cells |
| WO2022217869A1 (en) * | 2021-04-13 | 2022-10-20 | 广东唯泰生物科技有限公司 | Pariduval mesenchymal stem cell culture medium for treating tumors, and co-culture method |
| 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 |
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