CN106190969A - A kind of preparation method of decidua mescenchymal stem cell - Google Patents
A kind of preparation method of decidua mescenchymal stem cell Download PDFInfo
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Abstract
The invention discloses the preparation method of a kind of decidua mescenchymal stem cell, comprise the following steps: 1) sample and pre-treatment；2) cell separation；3) passage is cultivated；4) frozen.The method is the method preparing decidua mescenchymal stem cell from decidua tissue, on the basis of setting up ripe and safe and reliable isolation technics, obtain high-purity, high motility rate, pollution-free stem cell, and by its long term storage in liquid nitrogen, stem cell transplantation use can be done through recovery, and be expected to gradually to replace expensive, be difficult to find the bone marrow stem cell that coupling is harmonious.
Description
Technical field
The present invention relates to stem cells technology field, be specifically related to the preparation method of a kind of decidua mescenchymal stem cell.
Background technology
Stem cells technology and application thereof are considered as the human engineering health engineering of 21 century, and its research contents almost relates to
The biomedical sector of all life sciences, except transplanting in cell therapy, tissue/organ and play important work in gene therapy
With outward, also finding that the aspects such as new gene, gene function analysis, developmental biology model and new drug development produce material impact.
Refractory disease applied research clinically will be caused revolutionary advancement at medical domain by stem cells technology.The whole world is just at present
Stem cell in exploitation is mainly mescenchymal stem cell.
Mescenchymal stem cell is derived from the pluripotent stem cell in mature tissue, has height self-renewal capacity and multidirectional
Differentiation potential, can be divided into osteoblast, chondrocyte, hepatocyte, islet cells etc. under suitable inner or in vitro environment
Various kinds of cell.Additionally, mescenchymal stem cell has a low-down immunogenicity, thus become the new of cell transplantation and organizational project
Type seed cell, clinical research proves, mesenchymal stem cell transplantation has been successfully applied in graft versus host disease, apoplexy, the heart
In the disease treatments such as flesh infarction, idiopathic aplastic anemia, osteogenesis imperfecta, have broad application prospects.Mesenchyme
Source of human stem cell is widely, the most separable from the tissues such as bone marrow, fat, synovial membrane, muscle and amniotic fluid, Cord blood, Placenta Hominis
With prepare mescenchymal stem cell, but each there is certain problem, as apply most mesenchymal stem cells MSCs be subject to
Age effects differentiation capability, draws materials and is difficult to, and heteroplastic transplantation also causes the shortcomings such as immunoreation；There are certain human relations in embryonic stem cell
Reason problem；Fat, umbilical cord, placental samples gather and are all limited by many factors such as source, time, acquisition techniques.Therefore,
Explore that a kind of multiplication capacity is strong, immunogenicity is low, the mescenchymal stem cell of the restriction of amoral ethical issues is extremely urgent.
Endometrium mescenchymal stem cell, as a kind of special mescenchymal stem cell, derives from endometrial tissue, can
Separating from feminine menstrual blood and endometrial tissue and obtain, its quantity is 30 times of derived from bone marrow, has higher oneself more
New ability and multiplication capacity, differentiation potential is closer to embryonic stem cell.Studies have found that the regenerative cell (ERC) of endometrium no
Only there is the most every 24 hours the speed replicated once, and they produce the speed ratio of unique somatomedin from Cord blood
Stem cell to go up 100,000 times soon.The endometrium mescenchymal stem cell announced is basically separated from feminine menstrual blood, and it has
Gather convenient, not by advantages such as number of times are limited, but there is also the vulnerable to pollution that under ambient non-sterile, external individual's operation causes
Defect, so, it is possible to the source and the method that are separated to the endometrium mescenchymal stem cell of high-quality and guarantee aseptic safe are
Significantly.
Containing decidua tissue (i.e. endometrial tissue) in inside the material that the dilatation and curettage obtains, son can be obtained the most further
Endometrial stem cells.The dilatation and curettage is scraping endometrium or the operation of uterine cavity content, belongs to department of obstetrics and gynecology routine minor operation.Artificial abortion
The dilatation and curettage belongs to a kind of fractional curettage, is primarily adapted for use in First Trimester artificial abortion and incomplete abortion, placental retention etc. need emptying
Uterine cavity person.What artificial abortion's dilatation and curettage was obtained mainly include fine hair, decidua, endometrial tissue fragment, embryonal tissue and certain
Body fluid (blood) etc., wherein decidua tissue is that endometrial stroma is stimulated by decidualization inducible factor and breeds and be differentiated to form
A kind of particular tissues.Decidua can be divided into decidua basalis, decidua capsularis, decidua parietalis, and decidua basalis can form maternal placenta in the future, and bag is sloughed off
Film covers outside blastocyst, and decidua parietalis is close to cavity of uterus surface, either which kind of decidua, and either which rank the later stage grows
Section, is all diverse tissue with embryonal tissue, the most easily distinguishes.The dilatation and curettage of uterine sample of this patent is artificial abortion's dilatation and curettage of uterine
The decidua tissue that art is obtained, is the decidua tissue obtained after specialized clinician is selected, without fine hair etc. and embryo's group
Knitting relevant any tissue, and be gained in an aseptic environment, have good safety, it meets ethics, belongs to one
Plant " refuse " huge profit use, can be as a kind of source obtaining Endometrial stem cell.
Summary of the invention
In order to overcome the deficiencies in the prior art, the present invention provides the preparation of a kind of safe and reliable decidua mescenchymal stem cell
Method.
The purpose of the present invention realizes by the following technical solutions:
The preparation method of a kind of decidua mescenchymal stem cell, comprises the following steps:
1) sampling and pre-treatment: take decidua tissue, with brine, adds the collection liquid of 0.8-1.1 times of volume, low
Temperature storage；
2) cell separation: will be through step 1) process after sample antiseptic gauze filter, filtrate is carried out Viral diagnosis,
If filtrate testing result is negative, then the decidua tissue to corresponding filtration carries out following operation: washs 2 times with PBS, shreds,
Moving into centrifuge tube, add equal-volume 0.25% NTx enzymic digestion 30 minutes, rear addition equal-volume Chang complete medium is eventually
Only digestion, centrifugal, abandon supernatant, wash with PBS, add Chang complete medium re-suspended cell, obtain single cell suspension；
2) cell separation: will be through step 1) process after sample antiseptic gauze filter, filtrate is carried out Viral diagnosis,
If filtrate testing result is negative, then the tissue obtained after corresponding filtration is carried out following operation: wash 2 times with PBS, cut
Broken, move into centrifuge tube, add equal-volume 0.25% NTx enzymic digestion 30 minutes, rear addition equal-volume Chang complete medium
Terminate digestion, centrifugal, abandon supernatant, wash with PBS, add Chang complete medium re-suspended cell, obtain single cell suspension；
3) passage is cultivated: by step 2) single cell suspension of gained is inoculated in Chang complete medium cultivation 3-4
My god, change culture medium, the non-attached cell of reject, within the most every 2-3 days, change culture medium once, treat that cell growth reaches 80%-
During 90% fusion, it is the trypsin digestion and cell of 0.25% with weight percentage, adds Chang complete medium, and receive
Collection cell suspension；Cell suspension is placed in centrifuge tube, centrifugal, abandon supernatant, washed once with PBS, cultivate completely with Chang
The new suspension cell of basic weight；Cell after suspension is with 5000-6000/cm2Density carry out inoculate Secondary Culture, obtain purification
Decidua mescenchymal stem cell；
4) frozen: by step 3) decidua mescenchymal stem cell PBS flushing, add the trypsinization of 0.25%, add
Enter Chang complete medium resuspension, collect suspension cell, add frozen stock solution, be first placed in the program temperature reduction box equipped with isopropanol
After cooling, proceed to liquid nitrogen container and carry out frozen.
As preferably, step 1) in, described collection liquid is the DMEM culture medium comprising following components: ciprofloxacin, block that
Mycin and heparin.
As preferably, step 2) in, the concentration of described ciprofloxacin is 4mg/mL, and the concentration of described kanamycin is
10mg/mL, the concentration of described heparin is 800 units/L.
As preferably, step 2) in, described Chang complete medium consists of the following composition: MEM alpha culture medium,
Chang B, Chang C, Penicillin/Streptomycin, L-glutamine and ES-FBS.
As preferably, step 2) in, the Chang complete medium of every 102mL contains: the MEM alpha of 65mL cultivates
Base, 18mL Chang B, 2mL Chang C, 1mL Penicillin/Streptomycin, 1mL L-glutamine and
The ES-FBS of 15mL, wherein, the concentration of described L-glutamine is 200mM.
As preferably, step 2) in, the density of inoculation is 1 × 105-1×106/mL。
As preferably, step 3) in, Secondary Culture 37 DEG C, saturated humidity, volume fraction be the CO of 5%2In incubator
Carry out.
Compared to existing technology, the beneficial effects of the present invention is:
The invention provides a kind of method preparing decidua mescenchymal stem cell from decidua tissue, set up maturation
And on the basis of safe and reliable isolation technics, will obtain high-purity, high motility rate, pollution-free stem cell, and by its long term storage in
In liquid nitrogen, stem cell transplantation after recovery, can be used as, and have gradually replace expensive, be difficult to find the bone marrow that coupling is harmonious
The prospect of stem cell.
Accompanying drawing explanation
Fig. 1 is the cellular morphology figure during embodiment 1 subculture；
Fig. 2 is the growth curve chart of recovery cell；
Fig. 3 is the flow cytometer detection result figure before decidua mesenchymal stem cell cryopreserving；
Fig. 4 is the flow cytometer detection result figure after the recovery of decidua mescenchymal stem cell；
Fig. 5 is detection embodiment animal test results figure；
Fig. 6 is that P1 (A figure), P2 generation (B figure) cell of comparative example 2 shows with P1 (C figure), P2 generation (D figure) cell of embodiment 1
Micro-cyclogram.
Detailed description of the invention
Below, in conjunction with accompanying drawing and detailed description of the invention, the present invention is described further:
In detailed description below, such as non-specified otherwise, the test sample used, instrument, reagent etc. all can pass through
Commercially available approach obtains.
In detailed description below, the decidua tissue used takes from uterine cavity tissue, i.e. in the dilatation and curettage of uterine sample of the dilatation and curettage
All Informed Consent Form is signed with patient before gained decidua tissue, all dilatation and curettage of uterine sample collections and use.
The preparation method of the decidua mescenchymal stem cell that the present invention provides comprises the following steps:
1) sampling and pre-treatment: take decidua tissue, with brine, adds the collection liquid of 0.8-1.1 times of volume, low
Temperature storage；
2) cell separation: will be through step 1) process after sample antiseptic gauze filter, filtrate is carried out Viral diagnosis,
If filtrate testing result is negative, then its corresponding filtering residue is carried out following operation: wash 2 times with PBS, shred, move into centrifugal
Pipe, adds equal-volume 0.25% NTx enzymic digestion 30 minutes, and the rear equal-volume Chang complete medium that adds terminates digestion, from
The heart, abandons supernatant, washs with PBS, adds Chang complete medium re-suspended cell, obtains single cell suspension；
2) cell separation: will be through step 1) process after sample antiseptic gauze filter, filtrate is carried out Viral diagnosis,
If filtrate testing result is negative, then the tissue obtained after corresponding filtration is carried out following operation: wash 2 times with PBS, cut
Broken, move into centrifuge tube, add equal-volume 0.25% NTx enzymic digestion 30 minutes, rear addition equal-volume Chang complete medium
Terminate digestion, centrifugal, abandon supernatant, wash with PBS, add Chang complete medium re-suspended cell, obtain single cell suspension；
3) passage is cultivated: by step 2) single cell suspension of gained is inoculated in Chang complete medium cultivation 3-4
My god, change culture medium, the non-attached cell of reject, within the most every 2-3 days, change culture medium once, treat that cell growth reaches 80%-
During 90% fusion, it is the trypsin digestion and cell of 0.25% with weight percentage, adds Chang complete medium, and receive
Collection cell suspension；Cell suspension is placed in centrifuge tube, centrifugal, abandon supernatant, washed once with PBS, cultivate completely with Chang
The new suspension cell of basic weight；Cell after suspension is with 5000-6000/cm2Density carry out inoculate Secondary Culture, obtain purification
Decidua mescenchymal stem cell；
4) frozen: to treat step 3) cell, be decidua mescenchymal stem cell, with PBS rinse, add 0.25% pancreas egg
White enzymic digestion, adds Chang complete medium resuspension, collects suspension cell, adds frozen stock solution, is first placed in equipped with isopropanol
After program temperature reduction box cooling, proceed to liquid nitrogen container and carry out frozen.
Said method on the basis of setting up ripe and safe and reliable isolation technics, the decidua mescenchymal stem cell obtained
Its purity can reach more than 95%, its high motility rate, pollution-free stem cell, and by its long term storage in liquid nitrogen, can do through recovery
Stem cell transplantation use, and gradually replace expensive, be difficult to find the bone marrow stem cell that is harmonious of coupling.
In specific examples below, the liquid that gathers used is the DMEM culture medium comprising following components: ciprofloxacin, card
That mycin and heparin, its preparation example is as follows: add the ciprofloxacin of 2000mg, 500mg to the DMEM basal medium of 300mL
Kanamycin and 400 units heparin, add DMEM basal medium and be settled to 500mL, obtain collection liquid.This collection liquid is in 2-4 DEG C
Deposit under environment.
In specific examples below, used Chang complete medium consists of the following composition: MEM alpha culture medium,
Chang B, Chang C, Penicillin/Streptomycin, L-glutamine and ES-FBS, its preparation example is as follows:
Under aseptic condition, (MEM-alpha culture medium is bought from Invitrogen public to add the MEM alpha of 65mL in sterile chamber
Department), the Chang B base fluid of 18mL (buy from Irvine Scientific company), 2mL Chang C base fluid (buy from
Irvine Scientific company), the Penicillin/Streptomycin of 1mL (buy by penicillin/streptomycin sulfate
From Invitrogen company), (L-glutaminate, buys from Invitrogen company, and 15mL concentration is for the L-glutamine of 1mL
The ES-FBS (hyclone is bought from Invitrogen company) of 200mM, fully mixes.Before this Chang complete medium uses
Put 4 DEG C of refrigerators stand-by.
Embodiment 1
The preparation method of a kind of decidua mescenchymal stem cell, it is characterised in that comprise the following steps:
1) sampling and pre-treatment: take decidua tissue, with brine 3 times, the collection liquid of the body times volumes such as addition, 4-
10 DEG C of storages；
2) cell separation: will be through step 1) process after sample antiseptic gauze filter, filtrate is carried out Viral diagnosis,
If filtrate testing result is negative, then the tissue obtained after its corresponding filtration is carried out following operation: wash 2 times with PBS, use
Shears is by the tissue shear in filtering residue to 2mm3About size, move into centrifuge tube, add equal-volume 0.25% NTx enzyme in 37 DEG C
Digesting 30 minutes, the rear equal-volume Chang complete medium that adds terminates digestion, and 1200rpm/min is centrifuged 10min, abandons supernatant,
Wash 1 time with PBS, add Chang complete medium re-suspended cell, obtain single cell suspension；
In this step, described method for detecting virus includes but not limited to carry out Viral diagnosis by Elisa, detects project
Include but not limited to HBsAg, HCVAb, HIV-A, TP-PA, CMV-IgM, if positive findings, then terminate between whole storage decidua
The program of mesenchymal stem cells, decidua tissue sample is unsuitable for carrying out the preparation of follow-up decidua mescenchymal stem cell；
3) passage is cultivated: by step 2) single cell suspension of gained is inoculated in containing 10mL Chang complete medium
75cm2Culture bottle in, mix homogeneously, with Trypan Blue, number living cells；According to cell counts, adjust cell close
Degree is to 1 × 106/mL；In 37 DEG C, saturated humidity, volume fraction be the CO of 5%2Incubator is carried out cultivate 3 days；At aseptic condition
Lower replacing culture medium, the non-attached cell of reject, within later every 2 days, change culture medium once, treat that cell growth reaches 80%-90% and melts
During conjunction, being the trypsin digestion and cell of 0.25% with weight percentage, horizontal direction is jiggled, and makes pancreatin be paved with bottle
The end, after acting on 1 minute, under inverted microscope, observe digestion effect, after most cells (about 80%-90%) is reduced to circle,
Add 10ml Chang complete medium and terminate digestion, and collect cell suspension；
In this step, it is allowed to inoculum density be 1 × 105-1×106/mL；In this step, in incubation, it should protect
In card culture bottle, air communicates with air in incubator；
The cell suspension that will obtain, is placed in centrifuge tube, and 1000rpm is centrifuged 6min, abandons supernatant, washed once with PBS,
With Chang complete medium Eddy diffusion cell；The cell obtained by resuspension, with 5000-6000/cm2Density carry out
Pass on inoculation, 37 DEG C, saturated humidity, volume fraction be the CO of 5%2Incubator carries out Secondary Culture, is designated as P1；According to carefully
Intracellular growth situation, carries out full dose for every 2-3 days or half amount changes liquid, and during until cell reaches 80%～90% fusion again, cell spreads
At the bottom of full bottle, repeating above operation and pass on, be designated as P2, operation is passed on according to this, is decidua mescenchymal stem cell；
After 2-3 days full doses of cell primary cultivation change liquid, remove non-attached cell, Microscopic observation, it is seen that more several cells
The clone flocked together, cell proliferation is rapid, and its doubling time is about 24-36 hour, and after 2 days, cellular morphology is in the longest
Fusiformis, after cultivating 3-4 days, cell can get 80%-90% fusion, and cell is swirling distribution.Pass on rear cell complete in 24 hours
The most adherent, at the bottom of within 3-4 days, can being paved with bottle, continue to pass on.
Fig. 1 shows that the cellular morphology figure during subculture: A is original cuiture 3 days, changes the cell shape of 2 days after liquid
State figure；B is P1 for the figure of after passage 4 days, and cellular morphology is basically identical；C is P2 for the figure of after passage 3 days, and D is P7
For the figure of after passage 3 days, cell was swirling arrangement, and now cell purity is the highest.
These results suggest that, in terms of cellular morphology, the cell of separation and Culture of the present invention meets the feature of stem cell, cell
Multiplication capacity is stronger.
4) frozen: until step 3) P1 be paved with bottle for cell at the bottom of after, remove culture fluid, rinse with PBS, add 0.25%
Trypsin, keeps culture bottle to keep flat and make cell dissociation, adds Chang complete medium and makes cell suspension, through 1000rpm from
Heart 6min, abandons supernatant, collects suspension cell, Trypan Blue, calculates cell quantity and Cell viability, determine that Cell viability exists
More than 80%, add the cryopreservation tube containing equal-volume frozen stock solution, blow and beat gently with suction pipe and make cell uniform, seal, be positioned over
The good inside of rewarming drops in box equipped with the program of isopropanol, after placing 2h, subsequently relays in-80 DEG C of refrigerators in 4 DEG C of refrigerators
Realize slow cooling process, preserve in placing into liquid nitrogen storage；
In this step, frozen stock solution is that 20%DMSO with Chang complete medium is obtained by mixing according to the volume ratio of 1:9.Choosing
Select P1 and carry out frozen for cell, the initial condition of cell when both ensure that frozen, it is also possible to make cell amplification to sufficient amount level.
Each test tube is built-in is 1-2 × 10 containing density6The P1 of/mL is for cell 1mL.
Detection embodiment
1, recovery test
By embodiment 1 step 4) frozen after P1 for cell, from nitrogen storage tank take out, be immediately placed in 37 DEG C of water-baths
In, jog cryopreservation tube makes it all melt in 1 minute.
Aseptically, by soft for sample in cryopreservation tube sucking-off, the Chang complete medium containing 10ml is added
In 50ml sterile centrifugation tube, fully mix, at 4 DEG C, under conditions of 1000rpm centrifugal 6 minutes, centrifuge washing 2-3 time, remove and manage
Middle supernatant.Repetitive operation is once.Cell counting and motility rate analysis is carried out with trypan blue.According to 10000/cm2Density (profit
Use Chang complete medium) seed cells into 75cm2In culture bottle, put into 37 DEG C, saturated humidity, volume fraction be 5%
CO2Incubator is cultivated.According to cell growth status, within every 3～4 days, full dose changes liquid once, when reaching 80-90% fusion for cell,
It is the trypsinization of 0.25% by concentration, with 5000-6000/cm2Density passes on, and after recovery, cellular morphology is normal, increases
Growing speed not change, be passaged to about 30 generations, there is catabiosis in cell, finds out, carefully from the growth curve of Fig. 2 recovery cell
The doubling time of born of the same parents is maintained at 24-26 hour.
2, flow cytometer detection
Select step 3 in embodiment 1) the decidua mescenchymal stem cell in P5 generation that obtains, carry out flow cytometer detection.Each test tube
It is 5 × 10 built with concentration6The P5 of/mL is for cell 2mL.
1) prepared by cell suspension
With the 0.25% mass concentration trypsin of 1ml by cell dissociation, 500rpm, 5min are centrifugal, abandon supernatant.Add
PBS washes 2 times, then 2ml PBS re-suspended cell, and regulation cell concentration is 5 × 106/mL；
2) the fixing 15min of 4% paraformaldehyde (PBS preparation), then PBS washes 2 times (500rpm, centrifugal 5min)；
3) BSA of 5% fixes 40min.
4) every 10 are required according to streaming antibody (directly mark) dilution factor6Individual cell/200 μ L be separately added into 20 μ L antibody (CD29,
CD45, CD105, CD117, CD90, CD34, CD73, CD44, HLA-DR), lucifuge, hatch 30min for 37 DEG C；
5) wash one time with PBS, 1500rpm, centrifugal 5min, abandon supernatant, add 500 μ L PBS resuspended, machine examination in preparation
Survey.
6) flow cytometer is U.S. BD FAC.Antibody used from Becton Dickinson and Co. (Becton,
Dickinson&Company, family, Franklin, NJ).According to detection stem cell size feature utilize forward scattering light (FS),
Side scattered light (SS) finds cell population of interest.
The result of flow cytometer detection such as Fig. 3 and 4, Fig. 3 and 4 show, express CD73 before and after decidua mesenchymal stem cell cryopreserving,
CD90, CD105 percentage ratio is respectively 99.7%, 98%, 99.3%, cell CD73 after recovery, and CD90, CD105 percentage ratio is respectively
For: 98.62%, 99.51%, 92.68%.
3, tumorigenesis analysis
Selecting embodiment 1 step 3) gained cultivates the decidua mescenchymal stem cell in P5 generation and carries out tumorigenesis as follows and divide
Analysis.Because embryonic stem cell is generally of oncogenicity, the animal president tumor when animal experiment, the present embodiment carries out tumorigenesis analysis, with
Determine the safety of this decidua mescenchymal stem cell.1) bilayer soft agar Colony forming experiment
The double-deck semi-solid agar culture medium of preparation, adds bottom-layer agar (1.2%) and top-layer agar in 12 well culture plates
(0.7%).
Inoculation 200/mL unit decidua mescenchymal stem cell；37 DEG C, saturated humidity, 5%CO2Incubator is cultivated 14 days,
Observe clone's sum.
Matched group replaces decidua mescenchymal stem cell, inoculating cell consistent in density with stomach cancer cell line BGC 823.
Cell growth status after cultivating 14 days under equal conditions in vitro in Fig. 4, decidua mescenchymal stem cell group can not be
Breeding on soft agar, grow, do not form colony, arrow indication is single suspension cell, and experimental group tumor cell can infinitely increase
Grow breeding, form macroscopic bigger colony group.
2) zoopery
By 30, Balb/C nude mice is randomly divided into 3 groups: A group (n=10), B group (n=10), C group (n=10)；Carry out naked
Mus omoplate subcutaneous injection, A group injection 3 × 106The decidua mescenchymal stem cell of/200ul PBS dosage, B group injection 3 × 106/
The gastric cancer BGC823 cell strain of 200ul PBS dosage, C group injection 200ul PBS.Normal raising, solves in transplanting latter 2 months row
Cut open, observe and formed with or without tumor.
As seen from Figure 5, nude mice of control group tumor growth is obvious, to 60 days tumor substantially ulcerations, death of becoming thin, dissects
See splenomegaly；And decidua mescenchymal stem cell group has no that tumor nodule produces, dissect without obvious pathological changes, it can be seen that fill between decidua
Matter stem cell is without oncogenicity.This is contrary with the potential oncogenicity of embryonic stem cell, the decidua tissue of this decidua mescenchymal stem cell
There is not ethical issues in source.
Comparative example 1
As different from Example 1, comparative example 1 step 3) cryopreservation tube utilize programmed cooling instrument to carry out with borehole cooling journey
Sequence:
The first step, 4 DEG C, wait (after i.e. temperature is down to 4 DEG C, enter second step)；
Second step, 1.0 DEG C/per minute be down to-3.0 DEG C (casings)；
3rd step, 10.0 DEG C/min be down to-20.0 DEG C (casings)；
4th step, 1.0 DEG C/min be down to-40.0 DEG C (casings)；
5th step, 10.0 DEG C/min be down to-90.0 DEG C (casings).
Take P1 that comparative example 1 obtains for cell, be 1.5 × 10 for cell density in cryopreservation tube6The P1 of/mL for cell 1mL,
It is designated as T1；Example 1 step 3) P2 that obtains is for being 1.5 × 10 for cell density in cell, cryopreservation tube6In the P1 generation of/mL, is thin
Born of the same parents 1mL, is denoted as T2；Carry out cell counting and motility rate analysis according to the following steps:
1) configure under the conditions of the trypan blue mother solution of 4% mass fraction is stored in 4 DEG C, during use, be diluted to 0.4% with PBS.
2) frozen sample is taken out from nitrogen storage tank, recover according to the method for resuscitation in embodiment 2, centrifugal
Cell suspension is obtained after washing.
3) dyeing: cell suspension and 0.4% trypan blue solution are with 9:1 mix homogeneously.
4) counting: after dyeing terminates, be immediately placed in cell counter, respectively living cell counting and dead cell.
5) statistics cell viability: living cell rate (%)=total viable cell/(total viable cell+dead cell sum) ×
100%.
Cell counting and motility rate are analyzed as shown in table 1:
Table 1
Frozen cooling rate affect frozen rear cell survival rate, T2 be utilize the frozen stem cell of program temperature reduction box after proceed to again
Liquid nitrogen, Cell viability average out to more than 94.30% after recovery, T1 utilizes the cryopreservation methods that programmed cooling instrument program is arranged, recovery
Rear Cell viability average out to 94.11%.Although the stem cell motility rate that two kinds of methods are obtained is very nearly the same, but programmed cooling instrument instrument
Device itself is expensive, and frozen cost is high, and operating technology requires height, and program temperature reduction box interlayer is built with isopropanol, cooling
1 DEG C/min of speed, viability rate is high, and inoculated and cultured can quickly cover with, easy and simple to handle, adopts this method frozen
Mesenchymal stem cells, the most cost-effective ensures that stable frozen effect.
Comparative example 2
Use density－gradient centrifuga－tion method to prepare P1 from dilatation and curettage of uterine sample for decidua mescenchymal stem cell, specifically include following step
Rapid:
1) the filtrate 1500-2500g taking dilatation and curettage of uterine sample is centrifuged 8-12 minute, must be positioned at respectively under upper strata upper cleer and peaceful be positioned at
The blood of layer；
2) it is positive for taking part supernatant for Viral diagnosis, such as Viral diagnosis, then terminate whole operation；Such as Viral diagnosis
For feminine gender, then proceed following operation: after removing remaining supernatant, obtain blood；Use PBS dilute blood, PBS
With blood and to separate the volume ratio of liquid be 1.5-2.5:1；Blood after dilution is added on the pure man lymph separation liquid, the blood after dilution
Liquid and the volume ratio separating liquid are that 1.8-2.2:1600-1000g is centrifuged 12-18 minute, after being centrifuged, occur bright in centrifuge tube
Aobvious layering；Sucking-off is positioned at the mononuclear cell layer (tunica albuginea layer) of centre, with centrifugal after PBS washed cell (wash 2 times,
It is all that 400g is centrifuged 10 minutes every time), finally make single cell suspension with Chang complete medium.
3) by step 2) single cell suspension that obtains is inoculated in Chang complete medium, and cell-seeding-density is 1 ×
105-1×106/ mL, be placed in 37 DEG C, saturated humidity, volume fraction be the CO of 5%2Incubator is cultivated；Incubation time is
4-5 days；After the cultivation of 4-5 days terminates, change culture medium, the non-attached cell of reject；According to cell growth condition, every 3-4 days complete
Amount changes liquid once, when cell growth reaches 80%-90% fusion, is the trypsinization of 0.25% with weight percentage
Collect cell, then press 5000-6000/cm2Density passes on inoculated and cultured, and is designated as P1 generation；According to said method pass on 1 time,
P2 generation.Said process 37 DEG C, saturated humidity, volume fraction be the CO of 5%2Incubator is carried out.
With P1 and P2 of microscope observation comparative example 2 acquisition for cell, P1 and P2 itself and embodiment 1 obtained is for cell
Comparing, as shown in Figure 6, wherein A is the P1 generation of comparative example 2 to the cell micro observation figure of two kinds of method gained；B is comparative example
The P2 generation of 2；C is the P1 generation of embodiment 1；D is the P2 generation of embodiment 1.Contrast finds, the curettage tissue mentioned in the application method
In the stem cell that middle separation and Culture arrives either P1 generation, still P2 was higher for its purity, and in the dilatation and curettage of uterine filtrate in documents method
The stem cell that institute's separation and Culture obtains its contaminating cell contained identical algebraically when is more.On average, curettage tissue
P1 obtained by cultivation is 95% for cell purity, and P1 obtained by the cultivation of dilatation and curettage of uterine filtrate is about 90% for cell purity.
In sum, the inventive method successfully can separate from decidua tissue and obtain decidua mescenchymal stem cell, cell
Cultivate through amplification and purification and can cultivate many generations；Cell can be recovered continuation Secondary Culture after frozen, keeps stem cell special
Property.Through FCM analysis, cell high expressed CD29, CD105, CD90, CD73, CD44, not expression of HLA-DR, CD45,
CD117、CD34.Through tumorigenesis analysis, decidua mescenchymal stem cell is without oncogenicity.Use program temperature reduction box frozen decidua mesenchyme
Stem cells survival rate is high, easy and simple to handle, frozen effect stability.The present invention indicated above can effectively obtain high-quality decidua
Mescenchymal stem cell, the decidua mescenchymal stem cell separation and Culture storage method of the present invention is pratical and feasible, and has aseptic, peace
Entirely, purity height, low cost and other advantages.
It will be apparent to those skilled in the art that can technical scheme as described above and design, make other various
Corresponding change and deformation, and all these change and deformation all should belong to the protection domain of the claims in the present invention
Within.
Claims (7)
1. the preparation method of a decidua mescenchymal stem cell, it is characterised in that comprise the following steps:
1) sampling and pre-treatment: take decidua tissue, with brine, adds the collection liquid of 0.8-1.1 times of volume, and low temperature is store
Deposit；
2) cell separation: will be through step 1) process after sample antiseptic gauze filter, filtrate is carried out Viral diagnosis, if filter
Liquid testing result is negative, then the tissue obtained after corresponding filtration is carried out following operation: wash 2 times with PBS, shred, move
Entering centrifuge tube, add equal-volume 0.25% NTx enzymic digestion 30 minutes, the rear equal-volume Chang complete medium that adds terminates
Digestion, centrifugal, abandon supernatant, wash with PBS, add Chang complete medium re-suspended cell, obtain single cell suspension；
3) passage is cultivated: by step 2) single cell suspension of gained is inoculated in Chang complete medium cultivation 3-4 days,
Change culture medium, the non-attached cell of reject, within the most every 2-3 days, change culture medium once, treat that cell growth reaches 80%-90% and melts
During conjunction, it is the trypsin digestion and cell of 0.25% with weight percentage, adds Chang complete medium, and collect cell
Suspension；Cell suspension is placed in centrifuge tube, centrifugal, abandon supernatant, washed once with PBS, with Chang complete medium again
Suspension cell；Cell after suspension is with 5000-6000/cm2Density carry out inoculate Secondary Culture, obtain between the decidua of purification
Mesenchymal stem cells；
4) frozen: by step 3) decidua mescenchymal stem cell PBS flushing, add the trypsinization of 0.25%, add
Chang complete medium resuspension, collects suspension cell, adds frozen stock solution, is first placed in the program temperature reduction box equipped with isopropanol and drops
Wen Hou, then proceed to liquid nitrogen container and carry out frozen.
Method the most according to claim 1, it is characterised in that step 1) in, described collection liquid is to comprise following components
DMEM culture medium: ciprofloxacin, kanamycin and heparin.
Method the most according to claim 2, it is characterised in that step 2) in, the concentration of described ciprofloxacin is 4mg/mL,
The concentration of described kanamycin is 10mg/mL, and the concentration of described heparin is 800 units/L.
Method the most according to claim 1, it is characterised in that step 2) in, described Chang complete medium is by following one-tenth
Be grouped into: MEM alpha culture medium, Chang B, Chang C, Penicillin/Streptomycin, L-glutamine and
ES-FBS。
Method the most according to claim 4, it is characterised in that step 2) in, in the Chang complete medium of every 102mL
Contain: the MEM alpha culture medium of 65mL, the Penicillin/ of Chang C, 1mL of Chang B, 2mL of 18mL
The ES-FBS of L-glutamine and 15mL of Streptomycin, 1mL, wherein, the concentration of described L-glutamine is
200mM。
Method the most according to claim 1, it is characterised in that: step 2) in, the density of inoculation is 1 × 105-1×106/
mL。
Method the most according to claim 1, it is characterised in that: step 3) in, Secondary Culture 37 DEG C, saturated humidity, body
Fraction is the CO of 5%2Incubator is carried out.
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| CN108300689A (en) * | 2018-01-16 | 2018-07-20 | 佛山科学技术学院 | A method of separation and primary culture placental decidua vera mescenchymal stem cell |
| 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 |
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| 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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