CN106754637A - A kind of separating and extracting process of menses source temper endometrial stem cells - Google Patents

A kind of separating and extracting process of menses source temper endometrial stem cells Download PDF

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CN106754637A
CN106754637A CN201611024566.3A CN201611024566A CN106754637A CN 106754637 A CN106754637 A CN 106754637A CN 201611024566 A CN201611024566 A CN 201611024566A CN 106754637 A CN106754637 A CN 106754637A
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stem cells
separating
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林俊堂
刘彦礼
牛荣成
杨慈清
乔梁
管丽红
李小英
杨芬
孙钰椋
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Xinxiang Medical University
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Abstract

The present invention relates to a kind of separating and extracting process of menses source temper endometrial stem cells, it is comprised the following steps：1) learn from else's experience blood sample, add Sample preservation liquid to mix, be then centrifuged, collect sediment；2) by step 1) during the sediment that obtains adds erythrocyte cracked liquid, stood after mixing and cracked, then it is centrifuged, collect sediment；3) by step 2) sediment that obtains is resuspended with cell culture medium, then carries out cell culture, collects the cell that culture is obtained, as described menses source temper endometrial stem cells.Separating and extracting process of the present invention can quickly, efficiently separate extraction menses source temper endometrial stem cells, and simple to operate, cell high income, the damage of cytoactive is few.

Description

A kind of separating and extracting process of menses source temper endometrial stem cells
Technical field
The present invention relates to a kind of separating and extracting process of Endometrial stem cell, more particularly to a kind of menses source temper palace The separating and extracting process of inner membrance stem cell.
Background technology
In recent years, the rise of cell therapy excites the research boom to various stem cells, and be present in marrow, umbilical cord, Adult stem cell (Adult Stem Cells, ASCs) in various adult tissues such as Cord blood, fat, menses, not only overcomes Embryonic stem cell materials are difficult, differentiation is uncertain and the limitation such as ethics problem, while high efficiently multiplying ability and multidirectional point can be kept Change potential, for cell therapy provides good seed cell.
Uterus can increase through in the cycle in every month as the organ with superpower power of regeneration in human body, its endometrium About 5~7mm, and with this rate of rise, endometrium regular can come off and again in women's child-bearing period by 500 times or so Raw process, this not only shows menses source temper endometrial stem cells (Menstrual blood-derived Endometrium Stem Cells, MenESCs) abundance, while also imply MenESCs have stronger proliferation activity.As ASCs family The important member of race, MenESCs except expression CD29 (>99%), CD44 (>99%), CD73 (>99%), CD90 (>95%) and CD105(>99%) the ASCs surface marker beyond the region of objective existences such as, at the same express multi-lineage potential mark Oct-4, SSEA-4, Sox-2 and Nanog, implies that MenESCs has compared with differentiated potential.Additionally, compared to mesenchymal stem cells MSCs (BMSCs) and Cord blood The ASCs in source etc., MenESCs not only have more preferably proliferation activity, while also having stronger genetic stability (to reach 68 For when caryogram it is undistorted).Meanwhile, research shows, MenESCs does not express MHC Ι Ι receptoroids, trace expression MHC Ι receptoroids, secretly Show that MenESCs has relatively low immunogenicity.Therefore, MenESCs is once report, by its abundance, the separation of hurtless measure The advantage of the aspect such as mode and proliferation activity higher and differentiation potential, obtains extensive concern both domestic and external.Should in clinic On the premise of being ensured with the security of MenESCs, it is in diseases such as skeletonization, cardiac muscle, liver, endometrium and apoplexy Gratifying effect is illustrated in therapeutic process.
At present, the method for separation and Extraction MenESCs relies primarily on Ficoll lymphocyte separation mediums from through blood sample, Karyocyte in tunica albuginea layer is collected after density gradient centrifugation, further carries out follow-up cultivation after washing.Although using this Method can be successfully separated extraction MenESCs, but still have the following disadvantages：
First, Ficoll lymphocyte separation mediums used mainly have glucan and Sodium Amidotrizoate group when separating MenESCs Into its density is 1.077 ± 0.0001g/mL, and the mononuclearcell smaller than human lymphocyte density can be separated.Using process In, if the specimen amount of required separation is less, preferable mononuclear cell layer is formed using cell separation liquid energy, it is also convenient for collecting； But if needing, separate specimen amount is more, and not only separating liquid usage amount is big, and some red blood cells are remained suspended in point after centrifugation In chaotropic, to cause and mix more red blood cell in collection cell, and nucleus is also contaminated with red blood cell layer, cause cell yield compared with It is low.For donor can be collected into a menstrual cycle through blood sample more than 50mL, equal proportion be diluted to preservation liquid in Afterwards more than 100mL, a large amount of lymphocyte separation mediums can not only be consumed by density gradient centrifugation completely, increase the economy of operation And time cost, simultaneously because sample size is more, the yield of MenESCs will certainly be reduced, and the centrifugation of long period is to cell The damage of activity is more.
Secondly, it is complex compared to peripheral blood sample through blood sample, there is more MS and a large amount of blood clots, close Not only visible obvious tunica albuginea layer after degree gradient centrifugation, while the endometrium fragment that comes off of visible a large amount of suspensions.Experiment discovery, Suspend the endometrium fragment that comes off in contain substantial amounts of MenESCs, and the endometrium fragment that many comes off due to blood Blood clot is formed, therefore by the way that after density gradient separation the centrifuge tube bottom can be deposited in haemocyte, so as to reduce The yield of MenESCs.
The content of the invention
Based on this, it is an object of the present invention to provide a kind of separating and extracting process of menses source temper endometrial stem cells, It being capable of quickly and efficiently separation and Extraction menses source temper endometrial stem cells, and simple to operate, cell high income, cell The damage of activity is few.
A kind of separating and extracting process of menses source temper endometrial stem cells, comprises the following steps：
1) learn from else's experience blood sample, add Sample preservation liquid to mix, be then centrifuged, collect sediment；
2) by step 1) during the sediment that obtains adds erythrocyte cracked liquid, stood after mixing and cracked, then carry out Centrifugation, collects sediment；
3) by step 2) sediment that obtains is resuspended with cell culture medium, then carries out cell culture, and collect culture and obtain Cell, as described menses source temper endometrial stem cells (MenESCs).
Preferably, described Sample preservation liquid be containing 50~150U/mL penicillin, 0.8~1.2mg/mL streptomysins, 0.05~0.1mg/mL amphotericin Bs and 0.2~0.5mg/mL EDTA-Na2The PBS of (disodium ethylene diamine tetraacetate).
It is highly preferred that described Sample preservation liquid is to contain 100U/mL penicillin, 1mg/mL streptomysins, 0.1mg/mL two Property mycin B and 0.3mg/mL EDTA-Na2PBS.
Preferably, described erythrocyte cracked liquid is to contain 120~180mmol/L NH4Cl, 5~15mmol/L KHCO3 With 0.05~0.15mmol/L EDTA-Na2The aqueous solution.
It is highly preferred that described erythrocyte cracked liquid is to contain 150mmol/L NH4Cl、10mmol/L KHCO3With 0.1mmol/L EDTA-Na2The aqueous solution.
Preferably, the pH value of described erythrocyte cracked liquid is 7.2~7.4, preferably 7.2.
Preferably, described cell culture medium is the DMEM high glucose mediums containing 10% hyclone.
Preferably, in step 1) in, it is 1 through the consumption volume ratio of blood sample and Sample preservation liquid:1~1:3；What is be centrifuged turns Speed is 1200~1500rpm, and the time of centrifugation is 5~10 minutes.
Preferably, in step 2) in, erythrocyte cracked liquid and the consumption volume ratio through blood sample are 1:2~1:3；Cracking Time is 5~10 minutes；The rotating speed of centrifugation is 1200~1500rpm, and the time of centrifugation is 5~10 minutes.
Preferably, in step 3) in, the inoculum density of the cell culture is 5~10 × 104cells/cm2；Described is thin Born of the same parents' culture is in 37 DEG C, 5%CO2Cell culture incubator in cultivated.
Preferably, in step 3) in, described cell culture is by step 2) the sediment cell culture medium weight that obtains After outstanding, in 37 DEG C, 5%CO2Cell culture incubator in cultivate 2 days, wash not adherent cell off with PBS, add thin Born of the same parents' culture medium continues to cultivate, and hereafter changes a cell culture medium every 3 days, until cell fusion is to being passed on after 80%.
The separating and extracting process of menses source temper endometrial stem cells of the present invention, by the red blood cell described in use Lysate to being cracked through blood sample, can Reservations uterus inner membrance to greatest extent, the yield of target cell can be significantly improved； Meanwhile, the centrifugal rotational speed low (only needing 1200~1500rpm) of the separation and Extraction process, time are short (only needing 5~10 minutes), can Significantly reduce the damage to cytoactive.Separating and extracting process of the invention without lymphocyte separation medium, without using density Difference carrys out separation and Extraction karyocyte, it is to avoid use centrifugation under 1800rpm necessary to lymphocyte separation medium 20 minutes High speed centrifugation process, it is thus possible to effectively reduce the mechanical damage that high speed centrifugation is caused to cell.Separation and Extraction side of the invention Method is simple to operate, quickly and efficiently separation and Extraction can obtain described menses source temper endometrial stem cells, reduce it is economical and Time cost.
The Sample preservation liquid that separating and extracting process of the invention is used, by adding EDTA-Na2, can effectively play Anticoagulant effect, and being used in combination by penicillin, streptomysin and amphotericin B, can effectively play antibacterium, resist Fungi acts on, and does not interfere with the cytoactive of MenESCs.EDTA-Na2Can with through the calcium binding in blood sample into chelating Thing, makes calcium ion lose hemoglutination, so as to prevent blood clotting, it is to avoid MenESCs is separated through blood sample solidification causes shadow Ring；Penicillin, streptomysin have good sterilization, bacteriostasis, and amphotericin B is collected existing for position (vagina) to sample Fungi has stronger sterilization, bacteriostasis, and triple combination is used with stronger, broader spectrum of antibacterial action.
The erythrocyte cracked liquid that separating and extracting process of the invention is used, by NH4Cl、KHCO3And EDTA-Na2Conjunction Reason collocation, can form good hypotonic environment, crack seedless red blood cell water swelling, but influence to karyocyte compared with It is small, and splitting action is soft, can to greatest extent protect the activity of karyocyte.The erythrocyte cracked liquid and the consumption through blood sample Volume ratio is preferably 1:2~1:3, pyrolysis time is preferably 5~10 minutes, both can guarantee that effective cracking of cytode, and energy The activity of karyocyte is protected to greatest extent.In erythrocyte cracked liquid of the invention, NH4 +Ion cannot pass through cell membrane, Cl-From Son and HCO3 -Ion then can cross-film enter it is intracellular, improve intracellular ion concentration, hypotonic environment is formed in the solution, while EDTA is by chelating Mg2+And Ca2+Cell membrane stability can be reduced, because red blood cell lacks nucleus, its cytoskeletal structure phase It is more fragile for karyocyte, it is poor to expansion tolerance, by hypotonic effect water suction rupture in above-mentioned hypotonic environment, occur Haemolysis, and influence of the hypotonic environment to karyocytes such as MenESCs is smaller.Erythrocyte cracked liquid of the invention, by NH4Cl、 KHCO3And EDTA-Na2The strict control of concentration, ensure that red blood cell is thoroughly cracked, while ensureing to damage even cracking The karyocytes such as MenESCs.
In separating and extracting process of the invention, it is preferred to use the DMEM high glucose mediums containing 10% hyclone are used as cell Culture medium, DMEM culture mediums are more suitable for the growth of MenESCs cells, and sugar composition high can give the in-vitro multiplication of MenESCs cells Sufficient energy source is provided, and hyclone can provide abundant trophic factors for the growth of MenESCs cells, so that really Protect the pilot scale culture of MenESCs cells.
Brief description of the drawings
Fig. 1 is that MenESCs cells are schemed into fat, skeletonization, into chondrocyte induction differentiation detection.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.
Embodiment one：Collection is through blood sample
The preparation of Sample preservation liquid：PBS is taken, the penicillin of final concentration of 100U/mL, final concentration of is separately added into The EDTA-Na of the streptomysin of 1mg/mL, the amphotericin B of final concentration of 0.1mg/mL and final concentration of 0.3mg/mL2, it is fully mixed Close uniform.
Recruit the healthy women volunteer 10 of 3 days before the menstrual cycle, collect respectively its through blood sample each 5~ , then be pre-loaded with each being transferred to respectively through blood sample in the 50mL centrifuge tubes of 15mL Sample storage liquid by 15mL, tightens lid After turn upside down fully mix, obtain 10 parts of menses Sample storage liquid.Preserved at each menses Sample storage liquid is placed in into 4 DEG C, The separation and Extraction of MenESCs is completed in 48 hours.
Embodiment two：Using the method for the present invention (cells by red blood cell lysis method) separation and Extraction MenESCs
The preparation of erythrocyte cracked liquid：In purified water, the NH of final concentration of 150mmol/L is separately added into4Cl, final concentration It is the KHCO of 10mmol/L3And the EDTA-Na of final concentration of 0.1mmol/L2, after being sufficiently mixed uniformly, regulation pH value to 7.2.
Menses Sample storage liquid 10mL obtained in Example one, is transferred in 15mL sterile centrifugation tubes, with 1500rpm's Rotating speed is centrifuged 5 minutes, abandons supernatant, collects sediment.15mL sterile centrifugation tubes are taken, 10mL erythrocyte cracked liquids are added, will be heavy Starch is added in erythrocyte cracked liquid, after fully piping and druming is mixed, cracking 5 minutes is stood at room temperature, then turning with 1200rpm Speed centrifugation 5 minutes, abandons supernatant, collects karyocyte and endometrium sediment.
After karyocyte and endometrium sediment are washed into 2 times with PBS, 5mL is added to contain 10% tire ox blood Clear DMEM high glucose mediums are resuspended, cell density adjusted, with 5~10 × 104cells/cm2Inoculating cell blake bottle, 37 DEG C, 5%CO2Cell culture incubator in cultivate 2 days, then wash not adherent cell off with PBS, add culture medium after Continuous culture, hereafter changed a subculture, until then cell fusion is disappeared to 80% or so with 0.25% pancreatin every 3 days Change, passage.Collect the cell that culture is obtained, as described menses source temper endometrial stem cells (MenESCs).
Embodiment three：Using conventional method (density-gradient centrifugation method) separation and Extraction MenESCs
Menses Sample storage liquid 10mL obtained in Example one, is slowly added into 10mL Ficoll separation of lymphocytes In liquid, it is centrifuged 25 minutes with the rpm level of 1800rpm, obtains 4 layers of separating liquid, the careful separating liquid for removing the superiors, by Between tunica albuginea layer material and the endometrium fragment of suspension be transferred in 15mL sterile centrifugation tubes, washed with PBS 2 times Afterwards, add the DMEM high glucose mediums that 5mL contains 10% hyclone resuspended, adjust cell density, with 5~10 × 104cells/cm2Inoculating cell blake bottle, in 37 DEG C, 5%CO2Cell culture incubator in cultivate 2 days, then use PBS Wash not adherent cell off, add culture medium and continue to cultivate, hereafter a subculture was changed every 3 days, until cell fusion To 80% or so, digested with 0.25% pancreatin, passage.Collect the cell that culture is obtained, as described menses source temper palace Inner membrance stem cell (MenESCs).
Example IV：MenESCs cells yield is determined
Menses Sample storage liquid 20mL obtained in Example one, is equally divided into 2 parts, and every part of 10mL is respectively adopted this hair Bright cells by red blood cell lysis method (embodiment two) and traditional density-gradient centrifugation method (embodiment three) is separated and obtains primary menses Source temper endometrial stem cells (MenESCs), is then respectively adding the DMEM high glucose medium weights that 6mL contains 10% hyclone It is outstanding, and be inoculated in 6 porocyte culture plates, the cell that above two method is obtained respectively is inoculated with 3 holes.Tissue Culture Plate is placed in 37 DEG C, 5%CO2Cell culture incubator in cultivate 2 days, then wash not adherent cell off with PBS, add culture medium Continue to cultivate, continue to be fixed with 4% paraformaldehyde after cultivating 3 days.After through conventional violet staining, calculate pass through above-mentioned two respectively The primary MenESCs number of cell clones that the method for kind is obtained, measurement result is as shown in table 1 below.
The MenESC cell yield measurement results of table 1
Sample Number of cell clones
Cells by red blood cell lysis method 63 ± 11/hole
Density-gradient centrifugation method 38 ± 9/hole
Measurement result shows, the primary MenESCs cell clones digital display obtained using cells by red blood cell lysis method of the invention Write higher than the primary MenESCs number of cell clones obtained using traditional density gradient centrifugal process, it was demonstrated that red blood cell of the invention Cracking process can dramatically increase MenESCs cell yields, effectively improve through the utilization rate of blood sample.
Embodiment five：MenESCs surface marker analyte detections
The P3 that Example two, embodiment three are obtained respectively for MenESCs cell suspensions, adjustment cell density to 1 × 106cells/ml.Each 100 μ L of cell suspension of Example two, embodiment three, are separately added into monoclonal streaming antibody CD29- FITC、CD44-FITC、CD73-FITC、CD90-FITC、CD105-PE、CD34-FITC、CD45-FITC、HLA-ABC-FITC And HLA-DR-FITC, mix and be incubated 30 minutes after 4 DEG C of lucifuges, then washed with PBS 2 times, add culture medium weight It is outstanding.Then flow cytomery embodiment two, the cell surface marker expression of embodiment three, testing result are used respectively It is as shown in table 2 below.
The MenESCs surface marker testing results of table 2
Measurement result is shown, is obtained using cells by red blood cell lysis method of the invention and traditional density-gradient centrifugation method The expression of MenESCs cell surface markers, meets the requirement both at home and abroad to the expression of adult stem cell surface marker, shows Above two method can be successfully separated acquisition MenESCs, and therebetween in surface marker the positive expression rate without aobvious Write sex differernce.
Embodiment six：MenESCs cells break up detection into fat, skeletonization and into chondrocyte induction
The P3 that Example two, embodiment three are obtained respectively for MenESCs cell suspensions, with 1 × 104cells/cm2's Density is cultivated in being inoculated in 12 well culture plates respectively, treats cell fusion up to 50%, fat, skeletonization is carried out into respectively, into cartilage Induction and identification.
Adipogenic induction culture：Using DMEM high glucose mediums, 5% hyclone, 1 μm of ol/L dexamethasone, 200 μ are added Mol/L Indomethacins, 0.5mmol/L 3-isobutyl-1-methylxanthines, 10 μm of ol/L insulin；Changed once every 3 days Culture medium, Fiber differentiation 21 days.
Osteogenic induction culture：Using DMEM high glucose mediums, 5% hyclone, 0.1 μm of ol/L dexamethasone, 50 μ are added Mol/L ascoltins, 10mmol/L sodium β-glycerophosphates；A subculture, Fiber differentiation 21 days were changed every 3 days.
Into chondrocyte induction culture：Using DMEM high glucose mediums, add 5% hyclone, 0.1 μm of ol/L dexamethasone, 0.2mmol/L ascoltins, 1% Insulin-Transferrin-sodium selenate, 10ng/mL Transforming growth factor-β3s；Every 3 It changes a subculture, Fiber differentiation 21 days.
After Fiber differentiation terminates, washed with PBS respectively 2 times, then fix 30 minutes with 4% paraformaldehyde, then Washed with PBS 3 times.Oil red O, alizarin red, alcian blue are used respectively into fat, skeletonization, the cell into chondrocyte induction culture Dyeing, is subsequently placed in observation under inverted microscope, as a result as shown in Figure 1.
The testing result of Fig. 1 is shown, is obtained using cells by red blood cell lysis method of the invention and traditional density-gradient centrifugation method MenESCs cells, be respectively provided with multi-lineage potential, can under the induction of specific inducing culture into fat, skeletonization and into Cartilage direction breaks up, and meets requirement to adult stem cell multi-lineage potential both at home and abroad, shows that above two method can be into Work(is separated and obtains MenESCs cells.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously Can not therefore be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Scope.

Claims (9)

1. a kind of separating and extracting process of menses source temper endometrial stem cells, comprises the following steps：
1) learn from else's experience blood sample, add Sample preservation liquid to mix, be then centrifuged, collect sediment；
2) by step 1) during the sediment that obtains adds erythrocyte cracked liquid, stood after mixing and cracked, then it is centrifuged, Collect sediment；
3) by step 2) sediment that obtains is resuspended with cell culture medium, then carries out cell culture, and that collects that culture obtains is thin Born of the same parents, as described menses source temper endometrial stem cells.
2. the separating and extracting process of menses source temper endometrial stem cells according to claim 1, it is characterised in that：It is described Sample preservation liquid be mould containing 50~150U/mL penicillin, 0.8~1.2mg/mL streptomysins, 0.05~0.1mg/mL both sexes Plain B and 0.2~0.5mg/mL EDTA-Na2PBS.
3. the separating and extracting process of menses source temper endometrial stem cells according to claim 1, it is characterised in that：It is described Erythrocyte cracked liquid be to contain 120~180mmol/L NH4Cl, 5~15mmol/LKHCO3With 0.05~0.15mmol/L EDTA-Na2The aqueous solution.
4. the separating and extracting process of menses source temper endometrial stem cells according to claim 3, it is characterised in that：It is described Erythrocyte cracked liquid pH value be 7.2~7.4.
5. the separating and extracting process of menses source temper endometrial stem cells according to claim 1, it is characterised in that：It is described Cell culture medium be the DMEM high glucose mediums containing 10% hyclone.
6. the separating and extracting process of menses source temper endometrial stem cells according to claim 1, it is characterised in that：In step It is rapid 1) in, be 1 through the consumption volume ratio of blood sample and Sample preservation liquid:1~1:3；The rotating speed of centrifugation is 1200~1500rpm, The time of centrifugation is 5~10 minutes.
7. the separating and extracting process of menses source temper endometrial stem cells according to claim 1, it is characterised in that：In step It is rapid 2) in, erythrocyte cracked liquid with through blood sample consumption volume ratio be 1:2~1:3；The time of cracking is 5~10 minutes；From The rotating speed of the heart is 1200~1500rpm, and the time of centrifugation is 5~10 minutes.
8. the separating and extracting process of menses source temper endometrial stem cells according to claim 1, it is characterised in that：In step It is rapid 3) in, the inoculum density of the cell culture is 5~10 × 104cells/cm2；Described cell culture be 37 DEG C, 5% CO2Cell culture incubator in cultivated.
9. the separating and extracting process of menses source temper endometrial stem cells according to claim 8, it is characterised in that：In step It is rapid 3) in, described cell culture is by step 2) sediment that obtains with cell culture medium it is resuspended after, in 37 DEG C, 5%CO2's Cultivated 2 days in cell culture incubator, not adherent cell is washed off with PBS, added cell culture medium and continue to cultivate, hereafter A cell culture medium was changed every 3 days, until cell fusion is to being passed on after 80%.
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CN107988148A (en) * 2018-01-24 2018-05-04 北京臻溪谷医学研究中心（有限合伙） A kind of isolated culture method of Endometrial stem cell
CN110079496A (en) * 2018-01-25 2019-08-02 左凤琼 A kind of method of hypoxemia culture menses source Endometrial stem cell
CN109280635A (en) * 2018-11-23 2019-01-29 北京太东生物科技有限公司 A kind of Endometrial stem cell separation method
CN111040992A (en) * 2019-12-16 2020-04-21 杭州恩格生物医疗科技有限公司 Separation culture method of endometrial stem cells
CN111378618A (en) * 2020-03-26 2020-07-07 南京瑞沁生生物技术有限公司 Stem cell extraction and preparation method
CN112945667A (en) * 2021-02-01 2021-06-11 海南师范大学 Preparation method of blood single cell suspension for removing nucleated red blood cells
CN112945667B (en) * 2021-02-01 2024-03-26 海南师范大学 Preparation method of blood single-cell suspension for removing nucleated red blood cells
CN115386538A (en) * 2021-05-24 2022-11-25 上海长一干细胞研究中心有限公司 Method for separating, extracting and culturing endometrial stem cells from menstrual blood and special culture medium

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Application publication date: 20170531