CN102154202A - Method for storing endometrial stem cells - Google Patents
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- CN102154202A CN102154202A CN 201110085328 CN201110085328A CN102154202A CN 102154202 A CN102154202 A CN 102154202A CN 201110085328 CN201110085328 CN 201110085328 CN 201110085328 A CN201110085328 A CN 201110085328A CN 102154202 A CN102154202 A CN 102154202A
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Abstract
The invention discloses a method for storing endometrial stem cells, which comprises the following steps: 1) preparing a collection tool set and preparing a culture medium; 2) collecting menses; 3) performing a sterility test; and 4) subjecting endometrial stem cell to separation culture, namely, filtering mixed liquid, which is obtained by the step 2), in a collection tube 1, collecting single karyoplasts by using a density gradient centrifugation method, inoculating the obtained single karyoplasts in a Chang complete culture medium at an inoculation density of single karyoplasts of 1*10<5> to 1*10<6>/ml, and culturing in a culture tank which is at 37 DEG C and saturated humidity and contains CO2 at a volume percentage concentration of 5 percent; 5) amplifying and purifying cells; and 6) storing cells by freezing. When the method of the invention is used, a large number of high-purity target cells, namely endometrial stem cells, can be obtained.
Description
Technical field
The invention belongs to cellular segregation and cultivate and the preservation correlation technique, especially from menses, separate in utero intermembranous mesenchymal stem cells, the in utero foundation of film stem cell strain, and cultivate accordingly and the preservation technology.
Background technology
Stem cells technology is one of technology the most popular in the current life science, its research contents almost relates to the biomedical sector of all life sciences, except in cell therapy, tissue/organ transplanting and gene therapy, playing a significant role, also finding that aspects such as new gene, gene function analysis, developmental biology model and new drug development produce material impacts.In recent years, the research of stem cell has obtained important breakthrough, 1999 and 2000,10 big sciences that continuous 2 years of the most authoritative U.S. " Science " magazine in the world is classified stem cell and the Human Genome Project then as were broken through, especially in 1999 even classify stem-cell research as first.Stem cell causes revolutionary advancement at medical field probably, thereby has an immeasurable medical value, caused global extensive concern and research, U.S.'s " epoch " weekly thinks that the stem cell and the Human Genome Project will become the field that the new millennium has development and application prospect most simultaneously.
Mescenchymal stem cell (mesenchymal stem cells, be called for short MSCs) be the multipotential stem cell that derives from the mature tissue, having height self ability and multidirectional differentiation potential, is the cenospecies daughter cell of Transplanted cells and organizational project, has broad application prospects.Wherein, research MSCs the most widely is the mescenchymal stem cell that derives from marrow, reduces although its ethics problem is compared with embryonic stem cell greatly, and medullary cell must obtain by the approach of invading, and with advancing age, stem cell population significantly reduces.
As everyone knows, the endometrial cell cording has very strong self ability.In every month all can be in a organized way and the dramatic growth of blood vessel through the cycle, this propagation process can stop along with the end of menstrual cycle.The medium multinational scientist's of America and Japan current research is presented in the endometrial tissue that comes off with women's menses, has a considerable amount of mescenchymal stem cells (stroma stem cell)---film stem cell in utero, and quantity is 30 times of derived from bone marrow; These stem cell vigor are stronger, stronger self and multiplication capacity (the cultivation efficient as the myocardial cell is traditional 100 times of utilizing medullary cell to cultivate), and differentiation potential is more near embryonic stem cell.Have and discover that endometrial regenerative cell (ERC) not only has the surprising speed of duplicating once in almost per 24 hours, and they produce the speed of unique somatomedin, than big last 100,000 times of the stem cell that comes from Cord blood.If these stem cells are stored by scientific methods, women future in life, when in case major disease such as tumour or unexpected grievous injury take place, just can do the transplanting of autologous stem cells immediately, get well, alleviate oneself and household's economical load, alleviate burden on society, also might realize " youth is everlasting ", the wonderful dream of " beauty stays forever " that the women dreams of, have high-quality life.Women's in utero film stem cell not only can be used for me, also can be people's such as her children, relevant relatives (as hall cousin younger sister, even father and mother) clinical application.
Because domestic practiced only-child's policy in case suffer from blood system malignant disease and heredopathia, just can only adopt allogeneic stem cell transplantation, in utero the film stem cell is that allogeneic stem cell transplantation enriches best resource most.In case stored in utero film stem cell, just be equivalent to set up a prevention and treatment tumour and other relevant human bank that needs the stem-cell therapy disease for its whole family.The in utero film stem cell that stores also can be selected make stem cell transplantation usefulness for the whole nation and whole world patient, and progressively alternatively costs an arm and a leg, is difficult to find and joins the bone marrow stem cell that type is harmonious.The whole world waits for that the patient of stem cell transplantation is hundreds of millions of, only leukemia just reaches 3/100000ths, four at the sickness rate of China, promptly annual newly-increased about 40,000 patient, and the leukaemic of accumulative total has 4,000,000 approximately, wherein major part is children, and they are badly in need of giving treatment to by stem cell transplantation.
It is to obtain through initial gross separation, to remove most red corpuscle behind blood specimen that storage method through blood specimen is arranged at present, and it is frozen directly to add frozen storing liquid then.
Summary of the invention
The technical problem to be solved in the present invention provides the in utero method of film stem cell of a kind of storage.The present invention is directed to existing source of human stem cell difficulty or be subjected to problem such as ethics restriction, seeks a kind of wide material sources more efficiently and be not subjected to the separation of in utero film stem cell and the cultural method of ethics restriction.On this basis, increase and the purifying stem cell, and the stem cell of being cultivated is carried out form and Function Identification, thereby set up novel stem cell strain, and then fine stem cell resource is stored with stem cell characteristic.Superior part of the present invention is women's depleted menstrual blood wide material sources, and stem cell content is abundant, can guarantee the quality of the stem cell that is stored earlier by separation method efficiently.
In order to solve the problems of the technologies described above, the invention provides the in utero method of film stem cell of a kind of storage, may further comprise the steps successively:
1), preparation for acquiring suit and preparation substratum:
Collection tube 1: 20ml Hank ' s balanced salt solution (HBSS) is housed, and adds extremely following concentration of following composition: vancomycin (Vancomycin) 60～100 μ g/mL, Cephalexin Monohydrate Micro/Compacted (Claforan) 150～350 μ g/mL, kantlex (Kanamycin A Sulfate) 50～150 μ g/mL, gentamicin (Gentamycin) 80～160 μ g/mL, amphotericin B (Amphotericin B) 2～3 μ g/mL and 300～500 units heparin;
Collection tube 2: 10ml Hank ' s balanced salt solution (HBSS) is housed, and adds 150～250 units heparin;
Preparation Chang perfect medium: under aseptic condition, in sterile chamber, add 65mL MEM-alpha substratum (MEM alpha, Invitrogen company), 16～20mL Chang B base fluid (Irvine Scientific company), 1～3mL Chang C base fluid (Irvine Scientific company), 1～3mL penicillin or Streptomycin sulphate (Penicillin/Streptomycin, Invitrogen company, perhaps be penicillin vitriol or streptomycin sulfate), 1～3mL concentration is the L-glutaminate (L-glutamine of 200mM, Invitrogen company), foetal calf serum (the ES-FBS of 10～20mL, Invitrogen company), abundant mixing, it is stand-by to put 4 ℃ of refrigerators;
2), menses are collected:
Collect second or the 3rd day the menses that menstruation begins; The menses that in collection tube 1, add 15～20ml, the menses of adding 5～10ml in collection tube 2; And in 0～4 ℃ low temperature preservation down, described preservation period≤48h;
3), sterility test:
Collection tube 2 with after the centrifugal treating, is got supernatant liquor; Supernatant liquor is carried out anerobe and aerophil inspection according to the hemoculture method, and identify; If positive findings then finishes the in utero program of film stem cell of whole storage;
4), film stem cell separation and Culture in utero:
With step 2) mixed solution in the collection tube of gained 1 filters earlier, adopts density gradient centrifugation then, collects mononuclearcell;
The mononuclearcell of above-mentioned gained is inoculated in the Chang perfect medium, and the inoculum density of mononuclearcell is 1*10
5～1*10
6/ ml, placing 37 ℃, saturated humidity, volume fraction is that 5% CO2 incubator is cultivated;
5), cell amplification and purifying:
The mononuclearcell for the treatment of step 4) is changed the Chang perfect medium after being inoculated in 4～5 days of beginning cell cultures in the Chang perfect medium, and reject attached cell not; Per 3～4 days full doses are changed liquid once (can decide according to cell growth condition, full dose is changed liquid and promptly referred to change whole Chang perfect mediums) later on; Treating that cell growth reaches 80%～90% when merging, is the described trypsinase that uses 1ml in the cell of 0.25% the general per 1～20ml of trypsin with mass concentration) the digestion collecting cell, press then 5000-6000 individual/cm
2The density inoculation culture that goes down to posterity, and be designated as P1 generation;
6), cell cryopreservation:
After the P1 that treats step 5) is paved with container bottom for cell, with the quality percentage composition 0.25% tryptic digestion collecting cell; Frozen storing liquid re-suspended cell with precooling; Thereby the density that makes cell is 1～2*10
6/ ml; Frozen storing liquid is: DMSO (calbiochem company) preparation that adds 1 parts by volume in the Chang of 9 parts by volume perfect medium gets (being the Chang perfect medium that contains 10%DMSO);
Undertaken frozenly by the resuspended cell of frozen storing liquid with above-mentioned, frozen step is as follows:
The first step: 4 ℃, wait for (be after temperature is reduced to 4 ℃, entered for second step);
Second goes on foot 1.0 ℃/minute reduces to-3.0 ℃ (casing is spin manifold temperature);
The 3rd goes on foot 10.0 ℃/minute reduces to-20.0 ℃ (casing);
The 4th goes on foot 1.0 ℃/minute reduces to-40.0 ℃ (casing);
The 5th goes on foot 10.0 ℃/minute reduces to-90.0 ℃ (casing);
The 6th EOS;
Take out frozen sample, put into the nitrogen storage tank prolonged preservation.
As the in utero improvement of the method for film stem cell of storage of the present invention: the density gradient centrifugation in the step 4) is: will filter the mixed solution centrifugal (for example 1600g is centrifugal 10 minutes) of back gained, remove supernatant after, blood;
Use the PBS dilute blood, the volume ratio of PBS and blood is: 1.5～2.5: 1; Blood after the dilution is added on the human lymphocyte parting liquid, the volume ratio of blood after the dilution and human lymphocyte parting liquid is 1.5～2.5: 1, and centrifugal (for example 600g is centrifugal 15 minutes) are behind centrifugal the finishing, occur tangible layering in the centrifuge tube, the tunica albuginea layer is a mononuclearcell in the middle of the sucking-off.
In the present invention, the inner membrance stem cell separation and Culture of the sterility test of step 3) and step 4) can be carried out synchronously, and reason is: sterility test needs longer cycle, therefore, can not wait until that sterile culture goes out the separation and Culture of carrying out cell behind the result again.Monitoring result as step 3) is positive, then finishes the in utero program of film stem cell of whole storage.
Now the way of most of stem cell bank is after obtaining stem cell (or being mixed with its hetero-organization of stem cell, people's umbilical cord, amniotic fluid, peripheral blood etc.) from human body in the world, separate a little promptly carry out frozen, again will frozen cell recovery when needing by the time, cultivate and breed.
Superior part of the present invention is to obtain behind blood specimen from human body, through after the density gradient separation, remove most of red corpuscle, carry out the purifying amplification cultivation again, obtain the i.e. film stem cell in utero of purity is higher, quantity is bigger target cell, carry out frozenly again, so just guaranteed the quality of freeze-stored cell, and need to use this part cell later on, the incubation time after the recovery is also than the additive method much shorter.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is that the in utero film stem cell of cultivating different time among the embodiment 1 is amplified 100 times Photomicrograph;
A is P0 replacement liquid 4 days, 100X; B is 3 days generations of P1,100X; C is 3 days generations of P2,100X; D is 3 days generations of P7,100X; E is 3 days generations of P22,100X;
Fig. 2 is flow cytometry figure as a result among the embodiment 1.
Embodiment
Embodiment 1: store the in utero method of film stem cell, i.e. in utero separation, cultivation, amplification and frozen, the recovery of film stem cell of people; Carry out following steps successively:
1), preparation for acquiring suit and preparation substratum:
Collection tube 1: 20ml Hank ' s balanced salt solution (HBSS) is housed, and adds extremely following concentration of following composition: vancomycin (Vancomycin) 70 μ g/mL, Cephalexin Monohydrate Micro/Compacted (Claforan) 200 μ g/mL, kantlex (Kanamycin A Sulfate) 100 μ g/mL, gentamicin (Gentamycin) 140 μ g/mL, amphotericin B (Amphotericin B) 2.5 μ g/mL and add 400 units heparin.
Collection tube 2: 10ml HBSS (Hank ' s balanced salt solution) is housed, and adds 180 units heparin.
The beginning step 2) the collection menses before, above-mentioned collection tube 1 and collection tube 2 be deposited under 4 ℃ of environment.
Preparation Chang perfect medium: under aseptic condition, in sterile chamber, add 65mL MEM-alpha substratum (MEM alpha, Invitrogen company), 16mL Chang B base fluid (Irvine Scientific company), 2.5mL Chang C base fluid (Irvine Scientific company), 1mL penicillin (including the potassium salt of penicillin of 0.25g) as effective constituent, 1mL concentration is the L-glutaminate (L-glutamine of 200mM, Invitrogen company), 15mL ES-FBS (foetal calf serum, Invitrogen company), abundant mixing, it is stand-by to put 4 ℃ of refrigerators.
2), menses are collected:
Begin in volunteer (42 years old, signed Informed Consent Form) menstruation second day, take semi-crouch or full posture of squatting, after twice of menses cup doubling, put into intravaginal.Carefully take out after two hours, the menses 20ml in the menses cup is poured in the collection tube 1 (containing the described particular acquisition liquid of step 1) in advance); According to above step repetitive operation, in collection tube 2 (containing the described particular acquisition liquid of step 1) in advance), add the menses of 10ml again.
The collection tube 1 of gained and collection tube 2 are put into shaking table hatch, temperature is set to 4 ℃.
3), sterility test:
Shaking table is hatched after collection tube 2 after 24 hours carries out centrifugal treating (centrifugal 10 minutes of 1600g), supernatant liquor; Described supernatant liquor is carried out anerobe and aerophil inspection according to the hemoculture method of routine, and identify according to ordinary method.If positive findings then finishes the in utero program of film stem cell of whole storage.
4), film stem cell separation and Culture in utero:
Shaking table is hatched sample in the collection tube 1 after 24 hours with electronic pipettor piping and druming mixing, refilter clot and mucus in the sample, then be transferred in the 50ml centrifuge tube; The trim centrifuge tube, centrifugal 10 minutes of 1600g, the whizzer reduction of speed transfers to deep low gear; Density gradient centrifugation is separated mononuclearcell then, and is specific as follows:
At first remove the supernatant in the centrifuge tube; Note careful imbibition, do not upset cellular layer, cause extra cell loss;
Shifted the hemocyte of supernatant with PBS (volume ratio is about 2: 1) dilution by a certain percentage, electronic pipettor piping and druming mixing;
In Bechtop, get clean centrifuge tube, shift 15mL human lymphocyte parting liquid to every 50ml centrifuge tube;
Shop layer application of sample: adding on the good human lymphocyte parting liquid that the blood after will diluting is careful (moved during application of sample and wanted soft, avoid breaking up laminated fluid level or mix with laminated fluid level and influence separating effect), the volume ratio of blood after the dilution and human lymphocyte parting liquid is 2: 1;
The trim centrifuge tube, centrifugal 15 minutes of 600g, whizzer lifting speed will transfer to the lowest speed retaining, guarantees centrifugal stablizing, and layering is clear;
Extract mononuclearcell: behind centrifugal the finishing, occur tangible layering in the centrifuge tube, be followed successively by red corpuscle layer, GCL, separation liquid layer, monocyte (MNC) layer and plasma layer from down to up;
Sucking-off mononuclearcell layer: suction pipe or transfer pipet are directly stretched into the mononuclearcell layer, inhale this layer middle body cell earlier, cell around drawing again, it is soft that action is wanted, and guarantees this confluent monolayer cells not to be dispelled.Be creamy white if separate liquid layer and MNC layer, the interface is unclear, and prompting contains more mononuclearcell, takes the circumstances into consideration to draw and separates liquid layer, and speed is wanted slowly to guarantee not to be drawn to the red corpuscle layer;
Washed cell: the mononuclearcell of sucking-off is mixed with part cell parting liquid, needs to be washed with PBS; Add PBS with more than the cell dilution twice with electronic pipettor, mixing, centrifugal 10 minutes of 200g; Behind centrifugal the finishing, carefully take out centrifuge tube, remove supernatant; Repeat this step once;
Cell counting: with the abundant mixing of above gained cell, with the blue dyeing of platform dish, living cell counting;
Inoculating cell:, adjust cell density to 2*10 according to the cell counting result
5/ ml is inoculated in 75cm
2In the culturing bottle (the Chang perfect medium of interior dress 10ml).Certainly, the cell density during inoculation can adopt 1*10
5-1*10
6/ ml.
5), cell amplification and purifying:
Cell cultures is in 75cm
2In the culturing bottle (in establish that to contain density be 2*10
5The Chang perfect medium 10ml of/ml cell), place in the incubator, bottle cap is unscrewed half-turn, guarantees that bottle interior air communicates with air in the incubator, keeps 37 ℃ of incubators, saturated humidity, CO
2The state of volumetric concentration 5% begins to cultivate.
After 4 days, from CO
2Take out Tissue Culture Flask in the incubator, remove the substratum in the bottle under aseptic condition, reject is attached cell not, adds 10mL Chang perfect medium in culturing bottle, puts into CO once more
2Incubator is cultivated, and according to the cell growing state, every 3-4 days full dose is changed liquid once (being that full dose is changed the Chang perfect medium once).When treating that cell reaches the 80%-90% fusion, the tryptic digestion that in each culturing bottle, adds 1mL 0.25% (mass concentration), horizontal direction is jiggled, it is flat that pancreatin is paved with, act on after 1 minute, inverted microscope is observed the digestion effect down, treats that most of cell contracts to circle, adds 10mL Chang perfect medium again and stops digestion; Press 5000-6000/cm then
2The density inoculation culture that goes down to posterity, and be designated as P1 generation.In the culturing process that goes down to posterity, according to the cell growing state, carried out full dose in every 3-4 days or half amount is changed liquid, merge once more until cell, be paved with flatly, repeat above operation and go down to posterity, be designated as P2 generation, operation is gone down to posterity according to this.
After the former foster 3-4 days full doses of being commissioned to train of cell are changed liquid, remove not attached cell, mirror is observed down, as seen more several cell aggregations clone together, cell proliferation is rapid, and its doubling time is approximately 24-36 hour, and cellular form is even spindle shape after 2 days, cultivate after 4-5 days cell and can reach 80%-90% and merge, cell is the whirlpool shape and distributes.It is adherent fully in the cell 24 hours of back to go down to posterity, and at the bottom of can being paved with in 3-4 days bottle, continues to go down to posterity.After cultured cell in vitro was cultured to for 20 generations, the rate of propagation of cell obviously slowed down, and catabiosis appears in cell.
Fig. 1 has shown that former being commissioned to train of the cellular form figure that cultivates in the process that goes down to posterity: A supported 4 days, changes behind the liquid 2 days cellular form figure, and the visible cell form is more, circle, polygon, fusiformis etc. are arranged, and this moment, cell was more assorted; B be P1 for passage after 3 days figure, cellular form reaches unanimity; C be P2 for passage after 3 days figure, D be P7 for passage after 3 days figure, cell is the whirlpool shape to be arranged, this moment, cell purity was higher; E be P22 for passage after 3 days figure, cell density is lower, rate of propagation is slow, and presents catabiosis.
Above presentation of results, from cellular form, the cell of separation and Culture of the present invention meets the characteristics of stem cell, and the multiplication capacity of cell is stronger, and cell can be passaged to about 20 generations among the present invention.
6), cell cryopreservation
It is frozen to select P1 to carry out for cell, has both guaranteed the virgin state of cell when frozen, also can make cell amplification to the sufficient amount level.
Each is in vitro adorned, and to contain density be 1-2*10
6The P1 of/ml is for cell 1ml.
Treat P1 for cell grow to merge 80%-90% after, be 0.25% tryptic digestion collecting cell with the 1ml mass concentration.With a small amount of Chang perfect medium re-suspended cell, cell quantity and cell motility rate are calculated in the blue dyeing of platform dish, determine the cell motility rate more than 80%, and adjusting cell density with the Chang perfect medium is 2-4*10
6/ ml, the frozen storing liquid (10%DMSO) of adding equal-volume precooling (4 ℃); Be sub-packed in then in the frozen pipe of 2ml.The frozen storing liquid that adopts is that (that is, frozen precellular final concentration is 1-2*10 to the Chang perfect medium that contains 10%DMSO (calbiochem company)
6/ ml).
Frozen pipe is put into program control cooling instrument carry out precooling, begin frozen program.Frozen program is provided with as follows:
The first step: 4 ℃, wait for (be after temperature is reduced to 4 ℃, entered for second step);
Second goes on foot 1.0 ℃/minute reduces to-3.0 ℃ (casing);
The 3rd goes on foot 10.0 ℃/minute reduces to-20.0 ℃ (casing);
The 4th goes on foot 1.0 ℃/minute reduces to-40.0 ℃ (casing);
The 5th goes on foot 10.0 ℃/minute reduces to-90.0 ℃ (casing);
The 6th EOS;
Take out frozen sample, put into the nitrogen storage tank prolonged preservation.
Embodiment 2, cell recovery are cultivated
The frozen sample of the foregoing description 1 gained is taken out from nitrogen storage tank, put into 37 ℃ of water-baths immediately and thaw.When the ice-nucleus in observing frozen pipe has only the soya bean size, frozen pipe is taken out, put into mixture of ice and water at once.Under aseptic condition, with the soft sucking-off of sample in the frozen pipe, add in the aseptic centrifuge tube of 50mL, add the Chang perfect medium of 10mL more immediately, fully mixing washing under 4 ℃, the condition of 1000rpm centrifugal 10 minutes, is removed supernatant in the pipe.Repetitive operation once.Carry out cell counting and motility rate analysis with platform dish indigo plant.According to 10000/cm
2Density (utilizing the Chang perfect medium) seed cells into 75cm
2In the culturing bottle, put into 37 ℃, saturated humidity, volume fraction and be 5% CO
2Incubator is cultivated.According to the cell growing state, every 3-4 days full dose is changed liquid once, when treating that cell reaches 80%-90% and merges, is 0.25% tryptic digestion with concentration, with 5000-6000 individual/cm
2Density goes down to posterity, and recovery back cellular form is normal, and rate of propagation does not change, and is passaged to about 20 generations, and catabiosis appears in cell.
Embodiment 3. fluidic cells detect
The in utero intermembranous mesenchymal stem cells of selecting gained among the embodiment 1 to cultivate P5 generation is carried out streaming as follows and is detected.
Each in vitro is equipped with concentration is 5*10
6The P5 of/ml is for cell 2ml.
1) cell suspension preparation:
Pancreatin with 1ml 0.25% (mass concentration) gets off cell dissociation, 500rpm, and 5min is centrifugal, abandons supernatant.Add PBS and wash 2 times, 2ml PBS re-suspended cell is 5*10 thereby make cell concn then
6/ ml.
2) the fixing 15min of 4% Paraformaldehyde 96 (PBS preparation), PBS washes (1500rpm, centrifugal 5min) 2 times then.
3) the fixing 40min of 5% BSA.
4) require per 10 according to streaming antibody (straight mark) extent of dilution
6Individual cell/200ul adds 20ul antibody (CD39, CD44, CD90, CD34, HLA (ABC), HLA (DR-DP-DQ)) respectively, and lucifuge is hatched 30min for 37 ℃.
5) wash one time with PBS, 1500rpm, centrifugal 5min abandons supernatant, and it is resuspended to add 500ul PBS again, machine testing in the preparation.
6) flow cytometer is a U.S. Beckman Coulter company, model FC-500.Used antibody comes from Becton Dickinson and Co. (Becton, Dickinson ﹠amp; Company, the lake, Franklin, NJ).Utilize forward scatter light (FS), side scattered light (SS) to find cell population of interest according to the stem cell size characteristics that detect.
According to the fluorescein-labelled selection streaming sense channel of selected antibody, the fluorescence power of target cell is analyzed, calculate the positive per-cent of cell surface marker molecule.Specifically as shown in Figure 2.
From above embodiment as seen: the inventive method can successfully be separated in utero film stem cell of acquisition people from the women through blood sample, and cell can be cultured to for 20 generations through amplification and purifying cultivation; Cell continues to go down to posterity cultivation through recovering after frozen, keeps the stem cell form.Detect through fluidic cell, recovery cell expressing CD39, CD44, CD90 do not express CD34, HLA (ABC), HLA (DR-DP-DQ).Show that the present invention can set up in utero film stem cell strain of people, people of the present invention in utero film stem cell storage method is pratical and feasible, and has the advantage that other cell bank storage methods do not possess.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (2)
1. store the in utero method of film stem cell, it is characterized in that may further comprise the steps successively:
1), preparation for acquiring suit and preparation substratum:
Collection tube 1: 20ml Hank's balanced salt solution is housed, and adds extremely following concentration of following composition: vancomycin 60 ~ 100 μ g/mL, Cephalexin Monohydrate Micro/Compacted 150 ~ 350 μ g/mL, kantlex 50 ~ 150 μ g/mL, gentamicin 80 ~ 160 μ g/mL, amphotericin B 2 ~ 3 μ g/mL and 300 ~ 500 units heparin;
Collection tube 2: 10ml Hank's balanced salt solution is housed, and adds 150 ~ 250 units heparin;
Preparation Chang perfect medium: under aseptic condition, adding 65mL MEM-alpha substratum, 16 ~ 20mL Chang B base fluid, 1 ~ 3mL Chang C base fluid, 1 ~ 3mL penicillin or Streptomycin sulphate, 1 ~ 3mL concentration are the L-glutaminate of 200 mM, the foetal calf serum of 10 ~ 20mL in sterile chamber, abundant mixing, it is stand-by to put 4 ℃ of refrigerators;
2), menses are collected:
Collect second or the 3rd day the menses that menstruation begins; The menses that in collection tube 1, add 15 ~ 20ml, the menses of adding 5 ~ 10ml in collection tube 2; And in 0 ~ 4 ℃ low temperature preservation down, described preservation period≤48h;
3), sterility test:
Collection tube 2 with after the centrifugal treating, is got supernatant liquor; Described supernatant liquor is carried out anerobe and aerophil inspection according to the hemoculture method, and identify; If positive findings then finishes the in utero program of film stem cell of whole storage;
4), film stem cell separation and Culture in utero:
With step 2) mixed solution in the collection tube of gained 1 filters earlier, adopts density gradient centrifugation then, collects mononuclearcell;
The mononuclearcell of above-mentioned gained is inoculated in the Chang perfect medium, and the inoculum density of mononuclearcell is 1*10
5~ 1*10
6/ ml, placing 37 ℃, saturated humidity, volume fraction is 5% CO
2Cultivate in the incubator;
5), cell amplification and purifying:
The mononuclearcell for the treatment of step 4) is changed the Chang perfect medium after being inoculated in 4 ~ 5 days of beginning cell cultures in the Chang perfect medium, and reject attached cell not; Per 3 ~ 4 days full doses are changed liquid once later on; Treating that cell growth reaches 80% ~ 90% when merging, is 0.25% tryptic digestion collecting cell with mass concentration, press then 5000-6000 individual/cm
2The density inoculation culture that goes down to posterity, and be designated as P1 generation;
6), cell cryopreservation:
After the P1 that treats step 5) is paved with container bottom for cell, with the quality percentage composition 0.25% tryptic digestion collecting cell; Frozen storing liquid re-suspended cell with precooling; Thereby the density that makes cell is 1 ~ 2*10
6/ ml; Described frozen storing liquid is: the DMSO preparation that adds 1 parts by volume in the Chang of 9 parts by volume perfect medium gets;
Undertaken frozenly by the resuspended cell of frozen storing liquid with above-mentioned, frozen step is as follows:
The first step: 4 ℃, wait for;
Second goes on foot 1.0 ℃/minute reduces to-3.0 ℃;
The 3rd goes on foot 10.0 ℃/minute reduces to-20.0 ℃;
The 4th goes on foot 1.0 ℃/minute reduces to-40.0 ℃;
The 5th goes on foot 10.0 ℃/minute reduces to-90.0 ℃;
The 6th EOS;
Take out frozen sample, put into the nitrogen storage tank prolonged preservation.
2. storage according to claim 1 is the method for film stem cell in utero, it is characterized in that: the density gradient centrifugation in the described step 4) is: the mixed solution that will filter the back gained is centrifugal, behind the removal supernatant, gets blood;
Use the PBS dilute blood, the volume ratio of described PBS and blood is: 1.5 ~ 2.5:1; Blood after the dilution is added on the human lymphocyte parting liquid, and the volume ratio of blood after the dilution and human lymphocyte parting liquid is 1.5 ~ 2.5:1, and is centrifugal, behind centrifugal the finishing, occurs tangible layering in the centrifuge tube, and the tunica albuginea layer is a mononuclearcell in the middle of the sucking-off.
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| CN103374760A (en) * | 2012-04-19 | 2013-10-30 | 孙勇 | Construction of human endometrium (menstrual blood) stem cell bank |
| CN103849944A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Method for establishing uterine membrane stem cell bank |
| CN104560871A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | Culturing method of mesenchymal stem cells of menstrual blood |
| CN105112358A (en) * | 2015-09-08 | 2015-12-02 | 东莞赛尔生物科技有限公司 | Multifunctional menstrual blood stem cell culture method |
| EP3020276A1 (en) * | 2014-11-14 | 2016-05-18 | Milestone S.r.l. | Method and apparatus for collecting and preserving biological specimens |
| CN106256203A (en) * | 2016-08-12 | 2016-12-28 | 浙江译美生物科技有限公司 | A kind of stem cell cryopreserving system and using method thereof |
| CN106264688A (en) * | 2016-08-01 | 2017-01-04 | 北京臻惠康生物科技有限公司 | It is set with for Endometrium collection |
| CN106701660A (en) * | 2017-01-16 | 2017-05-24 | 广东万海细胞生物科技有限公司 | Endometrium stem cell serum-free medium |
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| CN103374760A (en) * | 2012-04-19 | 2013-10-30 | 孙勇 | Construction of human endometrium (menstrual blood) stem cell bank |
| CN103849944A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Method for establishing uterine membrane stem cell bank |
| EP3020276A1 (en) * | 2014-11-14 | 2016-05-18 | Milestone S.r.l. | Method and apparatus for collecting and preserving biological specimens |
| CN104560871A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | Culturing method of mesenchymal stem cells of menstrual blood |
| CN104560871B (en) * | 2014-12-29 | 2020-08-25 | 深圳市北科生物科技有限公司 | Method for culturing mesenchymal stem cells of menstrual blood |
| CN105112358B (en) * | 2015-09-08 | 2018-06-26 | 东莞赛尔生物科技有限公司 | Multifunctional menstrual blood stem cell culture method |
| CN105112358A (en) * | 2015-09-08 | 2015-12-02 | 东莞赛尔生物科技有限公司 | Multifunctional menstrual blood stem cell culture method |
| CN106264688A (en) * | 2016-08-01 | 2017-01-04 | 北京臻惠康生物科技有限公司 | It is set with for Endometrium collection |
| CN106264688B (en) * | 2016-08-01 | 2019-10-22 | 北京臻惠康生物科技有限公司 | It acquires for Endometrium with suit |
| CN106256203B (en) * | 2016-08-12 | 2019-06-28 | 浙江译美生物科技有限公司 | A kind of stem cell cryopreserving system and its application method |
| CN106256203A (en) * | 2016-08-12 | 2016-12-28 | 浙江译美生物科技有限公司 | A kind of stem cell cryopreserving system and using method thereof |
| CN106701660A (en) * | 2017-01-16 | 2017-05-24 | 广东万海细胞生物科技有限公司 | Endometrium stem cell serum-free medium |
| CN108220231A (en) * | 2018-01-23 | 2018-06-29 | 广州资生生物科技有限公司 | A kind of stem cell media and its preparation method and application |
| CN116004522A (en) * | 2023-01-31 | 2023-04-25 | 安徽易文赛生物技术有限公司 | Method and kit for separating, culturing and freezing regenerated cells of endometrium |
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Application publication date: 20110817 |