CN103555664A - Method for cultivating human placenta mesenchymal stem cells - Google Patents
Method for cultivating human placenta mesenchymal stem cells Download PDFInfo
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- CN103555664A CN103555664A CN201310579777.3A CN201310579777A CN103555664A CN 103555664 A CN103555664 A CN 103555664A CN 201310579777 A CN201310579777 A CN 201310579777A CN 103555664 A CN103555664 A CN 103555664A
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Abstract
The invention relates to a method for cultivating human placenta mesenchymal stem cells. The method comprises the steps of cleaning fresh human placenta tissue, and conserving in placenta protection fluid at 2-8 DEG C for further use; cleaning the human placenta tissue which is soaked in the placenta protection fluid for no more than 48 hours, sequentially immersing and cleaning the human placenta tissue in temporarily prepared sodium chloride solution respectively containing 2500-3500U/mL and 3500-4500U/mL of penicillin sodium and streptomycin sulfate as well as a metronidazole solution each milliliter of which contains 30-50U of injection amphotericin B, cleaning and adding to a sterile petri dish for further use, isolating and cultivating until cell fusion degree is 80-90%. The method can avoid potential danger caused by animal serum and animal-derived reagents; the method can be used for cultivating the human placenta mesenchymal stem cells within 48 hours, and has strong operability; the method can greatly reduce possibility of unqualified mesenchymal stem cell isolated culture caused by mold and anaerobic bacterium contamination.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of method of cultivator placenta mesenchyma stem cell.
Background technology
Stem cell is a kind of undifferentiated cell, there are two fundamental characteristics, the one, there is the of self-replication capacity, the 2nd, can be divided into more than one functioning cell, according to the size of differentiation potential, stem cell is generally divided into three classes, and the first kind is myeloid-lymphoid stem cell (totipotent stem cell), it can be divided into the totipotent cell that function is consistent, can grow for fetus; Equations of The Second Kind is pluripotent stem cell (pturipotent stem cel), and it can be divided into every kind of cell type in health, but can not form placenta or the necessary sustentacular tissue of fetation.Because the differentiation potential of pluripotent stem cell is not " all ", therefore this cell is not called to " all-round ", and they not embryos.The further specialization of pluripotent stem cell is multipotency (multiPotent) stem cell, and it is exclusively used in the cell of the specific germline that is divided into specific function specialization.Multipotential stem cell can be divided into the cell type containing in the tissue that they are derived from; For example blood stem cell is merely able to be divided into red blood cell, white cell and thrombocyte.Embryonic stem cell (Embryonic stem cell) has potential widely, can generate except all histocytes of extraplacental body; The 3rd class is that adult stem cell (aduit stem cell) is a kind of undifferentiated cell that will have lifelong self-replacation (identical copies) and self (self-renewal) ability, it is distributed in different tissues, and can develop and become various types of qualification cells.The classification of stem cell, is mainly divided into hemopoietic stem cell and non-hematopoietic stem cell.Hemopoietic stem cell is dominated the disease of blood, immunology, for example Cord blood of originating; Non-hematopoietic stem cell be take cytodifferentiation and reparation as main, can be applicable to the regenerative medicines such as apoplexy, diabetes, senile dementia, and there are umbilical cord, placenta, deciduous teeth etc. in source.
Mescenchymal stem cell (mesenchymal stem cell MSC) is the multipotential stem cell that can form various kinds of cell type.Mescenchymal stem cell (MSC) is found in marrow, also finds to be subsequently present in the Various Tissues perhaps of human body generation, growth course.In view of mescenchymal stem cell have multi-lineage potential, can hematopoiesis support and promote the features such as immunity is implanted, regulated to hemopoietic stem cell and separation and Culture is easy and simple to handle, just day by day receive people's concern.The range of application of mescenchymal stem cell is also more and more extensive.Carried out at present multinomial clinical application research both at home and abroad, principal disease type comprises osteoarthritis disease, liver cirrhosis, graft host rejection (GVHD), Spinal injury and degeneration nervous system disorders and diabetes etc.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method of cultivator placenta mesenchyma stem cell.
For solving technical problem of the present invention, the technical solution used in the present invention is as follows:
A method for cultivator placenta mesenchyma stem cell, is characterized in that: it comprises the steps:
(1) get the human placenta of fresh collection, clean, putting into 2-8 ℃ of placenta protection liquid saves backup, described placenta protection liquid adds benzylpenicillin sodium and Vetstrep to obtain in AIM-V substratum, and wherein benzylpenicillin sodium and the Vetstrep final concentration in umbilical cord protection liquid is respectively 100-200 U/mL and 100-200 U/mL;
(2) get to be immersed in placenta protection liquid and be no more than the human placenta of 48 hours, clean, put successively again and face the sodium chloride solution that is respectively 2500-3500 U/mL and 3500-4500 U/mL with the benzylpenicillin sodium of newly joining and Vetstrep content and every milliliter and embathe containing in the Metronidazule injection of 30-50 U amphotericin B, finally clean be again placed in disposable sterilized culture dish, carry out standby;
(3) human placenta of aseptic cleaning is cut into meat gruel shape, adds AIM-V substratum, put in separator tube and carry out separation with full-automatic separate tissue device, re-use aseptic strainer filtering, obtain placenta tissue cell suspension;
(4) placenta tissue cell suspension is carried out centrifugal, abandon supernatant, collect lower floor, be placed in culturing bottle, then add wherein serum free medium, the CO that to be placed in 36-38 ℃, saturated humidity, volume fraction be 5%
2in incubator, cultivate, to observing while forming a plurality of not of uniform size, cell island that cell quantity is intensive on bottle wall, obtain people's placenta mesenchyma stem cell primary cell mixed solution, then through centrifugal, can obtain people's placenta mesenchyma stem cell primary cell, then through going down to posterity, be cultured to cytogamy degree and reach 80-90% and get final product.
Press such scheme, placenta in described step (1) protection liquid joins in AIM-V substratum (treatment level) again and obtains after benzylpenicillin sodium for injection and streptomycin sulphate for injection are dissolved in advance; The sodium chloride solution that the middle benzylpenicillin sodium of described step (2) and Vetstrep content are respectively 2500-3500 U/mL and 3500-4500 U/mL is mixed to get benzylpenicillin sodium for injection and streptomycin sulphate for injection by a certain amount of adding in 0.9% sodium chloride injection; Described every milliliter of preparation containing the Metronidazule injection of 30-50 U amphotericin B be by amphotericin b for inj B first with after 0.9% sodium chloride injection dissolving, then be mixed to get with a certain amount of Metronidazule injection; Described people's placenta is respectively the sodium chloride solution of 2500-3500 U/mL and 3500-4500 U/mL and every milliliter at benzylpenicillin sodium and Vetstrep content and is 8-12min containing the time of embathing in the Metronidazule injection of 30-50 U amphotericin B.
Press such scheme, the cleaning in described step (1) and step (2) is 0.9% sodium chloride injection and cleans.
Press such scheme, the placenta tissue in described step (1) is after getting placenta and giving birth to, and gets the umbilical cord attachment area tissue block in 3-5cm around.
Press such scheme, described step (4), for cultivating after 5-6 days, is removed not adherent upper strata, then in culturing bottle, adds serum free medium continuation cultivation, and changed liquid once every 3-4 days, after cultivating 10-15 days, can obtain people's placenta mesenchyma stem cell primary cell;
Then the substratum in culturing bottle is shifted out completely, toward adding mass percent in culturing bottle, it is 0.25% the tryptic phosphate buffered saline buffer submergence of recombinant animal attached cell, put into 36～38 ℃ of incubator insulation digestion 7-9 minute, when observing the contracting of attached cell circle, and after at the bottom of having departed from bottle, add the serum free medium of Digestive system equal volume to stop digestion, then be transferred in centrifuge tube, the centrifugal supernatant of abandoning, blow the even cell that is deposited in centrifuge tube bottom, then according to 4000-6000 cell/cm
2be inoculated in culturing bottle, and supplement serum free medium in culturing bottle, put 36～38 ℃, saturated humidity, the CO that volume fraction is 5%
2in incubator, cultivate, every 3-4 days full dose is changed liquid once, merge each other to attached cell, and degrees of fusion reaches 80-90%, obtains people's placenta mesenchyma stem cell, whenever necessary, can repeat aforesaid operations and carry out manyly for cell cultures, increasing.
Press such scheme, described T25 culturing bottle can be placed people's placenta of being no more than 5g and cultivate through full-automatic separate tissue device processing, filtration, lower confluent monolayer cells after centrifugal; People's placenta that described T75 culturing bottle can be placed 10-15 g is cultivated through full-automatic separate tissue device processing, filtration, lower confluent monolayer cells after centrifugal; People's placenta that described T175 culturing bottle can be placed 30-35 g is cultivated through full-automatic separate tissue device processing, filtration, lower confluent monolayer cells after centrifugal;
The volume of the serum free medium needing in described T25 culturing bottle is 6-8mL; The volume of the serum free medium needing in described T75 culturing bottle is 20-25mL; The volume of the serum free medium needing in described T175 culturing bottle is 30-35mL.
First the separation preparation of people's placenta mesenchyma stem cell requires placenta tissue to have certain activity, is generally the activity that guarantees placenta tissue, need to process in time placenta, and this has also brought obstacle to practical application.The present invention preserves by the placenta of fresh collection being placed in to placenta protection liquid, can reach the effect to microbial disinfection residual in placenta, can maintain again the active object of placenta tissue.Use 2-8 ℃ of placenta of preserving of placenta protection liquid of the present invention in 48 hours, all to can be used for the cultivation of placenta mesenchyma stem cell, this produces to actual preparation and has brought great operability.
Except this, the present invention is when carrying out aseptic cleaning to placenta, except using the sodium chloride solution of benzylpenicillin sodium and Vetstrep to carry out aseptically process, the present invention is the special self-pollution feature in conjunction with placenta also, especially people's placenta that natural labor is collected is subject to the problem of anerobe and mould contamination, added especially in the Metronidazule injection of amphotericin b for inj B and carried out aseptically process, and can obviously improve thus the success ratio that people's placenta mesenchyma stem cell is cultivated.Wherein, Metronidazule injection mainly can be used for the treatment of anti anaerobic bacteria infection, anti-trichomonal, amphotericin b for inj B is polyenoid class antifungal drug, thereby its eubolism that can destroy the fungal cells such as Cryptococcus neoformans, Blastomyces dermatitidis, histoplasma capsulatum, Coccidioides, Sporothrix, Candida suppresses its growth.But amphotericin b for inj B has certain toxicity to cell, the present invention, through repeatedly experimental studies have found that, by controlling the concentration of amphotericin B, can reach the active object that it is played a role but do not affect placenta mesenchyma stem cell.
Beneficial effect of the present invention:
(1) the present invention is omnidistance uses the reagent of serum-free animal origin-free and utilizes full-automatic tissue processor, substitute that traditional people's placenta mesenchyma stem cell uses in cultivating foetal calf serum, trypsinase and the collagenase of animal-origin, the potential danger that can avoid the reagent of animal serum and animal-origin to bring in clinical application;
(2) after collection, put into placenta protection liquid, can play the effect that keeps in vitro tissue activity, make manufacture have stronger operability, in 48 hours, separated preparation, still can isolate the placenta mesenchyma stem cell that activity is good;
(3) placenta cleaning process is except using the mycillin of wide spectrum to carry out aseptically process, add amphotericin B and Metronidazule injection, very effective to processing with the umbilical cord of mould, anerobe in birth process, can greatly reduce the underproof probability of mescenchymal stem cell separation and Culture causing because of mould, anerobe pollution.
Embodiment
Embodiment 1
1. after placenta is given birth to; get the umbilical cord attachment area tissue block in 5cm around; after cleaning, the sodium chloride injection that is 9g/L through 0.9% sodium chloride injection joins in placenta protection liquid in 2-8 ℃ of preservation; described placenta protection liquid joins in AIM-V substratum (treatment level) after benzylpenicillin sodium for injection and streptomycin sulphate for injection are first dissolved with a small amount of AIM-V substratum respectively, and wherein benzylpenicillin sodium and Vetstrep protect the final concentration in liquid to be respectively 200U/mL and 200U/mL at placenta.So, placenta is put in placenta protection liquid and is preserved in 2-8 ℃ of preservation, can make placenta in 48 hours, all can be used for separation under the protection of placenta protection liquid and prepare mescenchymal stem cell.
2. the aseptic cleaning of people's placenta
With aseptic nipper, from placenta preservation liquid, take out people's placenta and put into sterilising vessel, add 250mL 0.9% sodium chloride injection, with 8cm aseptic nipper, people's placenta is shaken to rinsing once back and forth.After with aseptic nipper, people's placenta is transferred in another sterilising vessel again, add 250mL to face the sodium chloride solution (dense dual anti-solution) that is respectively 3200U/mL, 4000U/mL with the benzylpenicillin sodium of newly joining and Vetstrep, every 1-2 minute, with aseptic nipper, people's placenta is shaken several times back and forth, total immersion is washed 10 minutes.With aseptic nipper, people's placenta is picked up again, transfer to the Metronidazule injection that adds every milliliter of amphotericin b for inj B of Han50 unit in sterilising vessel, with same method, embathe 10 minutes.Embathe and with 8cm aseptic nipper, people's placenta is picked up afterwards, then put in another sterilising vessel, add 0.9% sodium chloride injection, and people's placenta is shaken back and forth and cleaned three times, the people's placenta after cleaning is placed in disposable sterilized culture dish.
Above-mentioned dense dual anti-solution is for newly joining before use, and concrete compound method is: get each of the benzylpenicillin sodium for injection of 80Wan unit and the streptomycin sulphate for injection of 100Wan unit and join in 250mL 0.9% sodium chloride injection and obtain.
Above-mentioned every milliliter of preparation containing the Metronidazule injection of 50U amphotericin B: get mono-of the amphotericin b for inj B of 2.5Wan unit, add 5mL 0.9% sodium chloride injection to dissolve, then get and mix in aforementioned solution 1mL and 100mL Metronidazule injection and obtain.
To finally clean the 0.9% sodium chloride injection draw samples that placenta uses and carry out the detection of microorganism situation through membrane-filter procedure: through 14 days culture sample, have no bacterium, mould-growth.
3. the separation of people's placenta stem-cell preparation
People's placenta that aseptically process in disposable sterilized culture dish is crossed is removed blood vessel and mucous membrane wherein with 5cm aseptic nipper and scissors, then by 5cm sterile scissors, is cut into meat gruel shape.Get 10g at every turn and put C pipe (the full-automatic supporting separator tube of tissue processor), add 8mLAIM-V substratum, through full-automatic separate tissue device, process as under full-automatic tissue processor (C-01) program, stir 2 times and process, be then transferred in 50mL centrifuge tube; Use the same method and process remaining meat gruel shape human placenta, until whole separated complete all people's placenta tissue.
Then use after the aseptic strainer filtering of the disposable use of 100 μ m, obtain placenta tissue cell suspension, by placenta tissue cell suspension centrifugal 15min under 300g, abandon supernatant, collect lower confluent monolayer cells, be placed in respectively 2 T25 culturing bottles, then add wherein after serum free medium, culturing bottle is put to the CO that 36～38 ℃, saturated humidity, volume fraction are 5%
2in incubator, cultivate.The quantity of culturing bottle can be selected according to the weight of processing placenta, and people's placenta that the general reducible placement of T25 culturing bottle is no more than 5g is through automatically separate tissue device processing, filtration, cell after centrifugal.The substratum that T25 culturing bottle adds is 6-8mL.
4. the former culture of people's placenta mesenchyma stem cell
Cultivate after 5 days, not adherent upper strata is removed, then toward adding serum free medium in culturing bottle, proceed to cultivate, and changed liquid once every 3-4 days, after about 10-15 days, available inverted microscope has been observed attached cell growth, then continue to cultivate, can observe attached cell and be proliferated into gradually larger cell colony.When observing Tissue Culture Flask wall and form a plurality of not of uniform size, cell island that cell quantity is intensive, obtain people's placenta mesenchyma stem cell primary cell mixed solution, then through centrifugal, can obtain people's placenta mesenchyma stem cell primary cell.
5. people's placenta mesenchyma stem cell goes down to posterity
Substratum in culturing bottle is shifted out completely, toward adding mass percent in culturing bottle, be that (T25 adds 3mL for 0.25% the tryptic phosphate buffered saline buffer of recombinant animal, T75 adds 4mL, T175 adds 6mL), then put into 36～38 ℃ of about 7-9 minute of incubator insulation digestion, in this process, under inverted microscope, observe the contracting of attached cell circle, and after at the bottom of cell has departed from bottle, add the serum free medium of Digestive system equal volume to stop digestion, then be transferred in centrifuge tube, with expecting blue dyeing meter cytoactive with tally counting and tongue thereof on binocular microscope.Then centrifuge tube is put to centrifugal 5min under 300g condition, abandoned supernatant after centrifugal, with Pasteur's pipe, blow gently the even cell that is deposited in centrifuge tube bottom, then according to 4000-6000 cell count/cm
2be inoculated in culturing bottle, and carry out mark, in culturing bottle, supplement serum free medium, put 36～38 ℃, saturated humidity, the CO that volume fraction is 5%
2in incubator, cultivate, every 3-4 days full dose is changed liquid once.After cultivating 24h, passage cell starts adherent, becomes single dispersion long strip shape cell, changes liquid can remove not attached cell after 3-4 days by full dose, and now cell is the spindle shape cell that degrees of fusion reaches 50% left and right.After approximately 6 days, cell based instinct reaches more than 80% fusion, gets final product to obtain mescenchymal stem cell, then can repeat as required aforesaid operations and carry out P2, P3, P4 ... go down to posterity.
Embodiment 2
1. after placenta is given birth to; get the umbilical cord attachment area tissue block in 3cm around; after cleaning, 0.9% sodium chloride injection joins in placenta protection liquid in 2-8 ℃ of preservation; described placenta protection liquid is to add benzylpenicillin sodium and Vetstrep in AIM-V substratum (treatment level), and wherein benzylpenicillin sodium and the Vetstrep final concentration in placenta protection liquid is respectively 100U/mL and 100U/mL.So, placenta is put in placenta protection liquid and is preserved in 2-8 ℃ of preservation, can make placenta in 48 hours, all can be used for separation under the protection of placenta protection liquid and prepare mescenchymal stem cell.
2. the aseptic cleaning of people's placenta
With aseptic nipper, from placenta preservation liquid, take out people's placenta and put into sterilising vessel, add 0.9% sodium chloride injection, with 8cm aseptic nipper, people's placenta is shaken to rinsing once back and forth.After with aseptic nipper, people's placenta is transferred in another sterilising vessel again, add and face the sodium chloride solution (dense dual anti-solution) that is respectively 2500U/mL, 3500U/mL with the benzylpenicillin sodium of newly joining and Vetstrep, every 1-2 minute, with aseptic nipper, people's placenta is shaken several times back and forth, total immersion is washed 8 minutes.With aseptic nipper, people's placenta is picked up again, transfer in sterilising vessel and add every milliliter containing the Metronidazule injection of 30U amphotericin B, with same method, embathe 8 minutes.Embathe and with 8cm aseptic nipper, people's placenta is picked up afterwards, then put in another sterilising vessel, add 0.9% sodium chloride injection, and people's placenta is shaken back and forth and cleaned three times, the people's placenta after cleaning is placed in disposable sterilized culture dish.
Above-mentioned dense dual anti-solution is for newly joining before use, and it is preparation and every milliliter of compound method reference example 1 containing the Metronidazule injection of 30U amphotericin b for inj B specifically.
To finally clean the 0.9% sodium chloride injection draw samples that placenta uses and carry out the detection of microorganism situation through membrane-filter procedure: through 14 days culture sample, have no bacterium, mould-growth.
3. the separation of people's placenta stem-cell preparation
People's placenta that aseptically process in disposable sterilized culture dish is crossed is removed blood vessel and mucous membrane wherein with 5cm aseptic nipper and scissors, then by 5cm sterile scissors, is cut into meat gruel shape.Get 10g at every turn and put C pipe (the full-automatic supporting separator tube of tissue processor), add 8mL AIM-V substratum, through full-automatic separate tissue device, process, be then transferred in 50mL centrifuge tube; Use the same method and process remaining meat gruel shape human placenta, until whole separated complete all people's placenta tissue.
Then use after the aseptic strainer filtering of disposable use, obtain placenta tissue cell suspension, placenta tissue cell suspension is centrifugal, abandon supernatant, collect lower confluent monolayer cells, be placed in culturing bottle, then add wherein after serum free medium, culturing bottle is put to the CO that 36～38 ℃, saturated humidity, volume fraction are 5%
2in incubator, cultivate.
4. the former culture of people's placenta mesenchyma stem cell
Cultivate after 5-6 days, not adherent upper strata is removed, then in culturing bottle, adding serum free medium proceeds to cultivate, and changed liquid once every 3-4 days, after about 10-15 days, available inverted microscope has been observed attached cell growth, then continue to cultivate, can observe attached cell and be proliferated into gradually larger cell colony.When observing Tissue Culture Flask wall and form a plurality of not of uniform size, cell island that cell quantity is intensive, obtain people's placenta mesenchyma stem cell primary cell mixed solution, then through centrifugal, can obtain people's placenta mesenchyma stem cell primary cell.
5. people's placenta mesenchyma stem cell goes down to posterity
Substratum in culturing bottle is shifted out completely, toward adding mass percent in culturing bottle, it is 0.25% the tryptic phosphate buffered saline buffer of recombinant animal, then put into 36～38 ℃ of about 7-9 minute of incubator insulation digestion, in this process, under inverted microscope, observe the contracting of attached cell circle, and after at the bottom of cell has departed from bottle, add the serum free medium of Digestive system equal volume to stop digestion, then be transferred in centrifuge tube, with expecting blue dyeing meter cytoactive with tally counting and tongue on binocular microscope.Then centrifuge tube is centrifugal, abandon supernatant, with Pasteur's pipe, blow gently the even cell that is deposited in centrifuge tube bottom, then according to 4000-6000 cell count/cm
2be inoculated in culturing bottle, and carry out mark, in culturing bottle, supplement serum free medium, put 36～38 ℃, saturated humidity, the CO that volume fraction is 5%
2in incubator, cultivate, every 3-4 days full dose is changed liquid once.After cultivating 24h, passage cell starts adherent, becomes single dispersion long strip shape cell, changes liquid can remove not attached cell after 3-4 days by full dose, and now cell is the spindle shape cell that degrees of fusion reaches 50% left and right.After approximately 6 days, cell based instinct reaches more than 80% fusion, gets final product to obtain mescenchymal stem cell, then can repeat as required aforesaid operations and carry out P2, P3, P4 ... go down to posterity.
The mescenchymal stem cell that embodiment 1 and 2 is obtained carries out fluidic cell detection, detection method and the results are shown in down:
Experimental principle: utilize Flow Cytometry to detect fluorescent-labeled antibody.According to antigen-antibody combination principle, with the antibody of specific fluorescent element mark, the known cell that carries corresponding antigens is dyeed.Cell through fluorescein labelled antibody dyeing can be identified by flow cytometer, and according to the intensity of the entrained fluorescein of known cell, traget antibody is carried out qualitative, quantitative analysis.
Detecting step:
(1) get 5 streaming pipes, be labeled as respectively 1. 2. 3. 90/34/14 4. DR/105/19 5. 45/34 of FITC/PE sample tube of FITC/PE/APC of control tube.
(2) each streaming pipe adds respectively passage cell (number of nucleated cells 2-5 * 10 of same amount
5individual), can not adhere to tube wall.
(3) 1. control tube adds Mouse IgG2a-PE, Mouse IgG1-FITC, Mouse IgG1-APC 5 ul respectively, mixes; 2. control tube adds Mouse IgG2a-PE, Mouse IgG1-FITC 5 ul respectively, mixes; 3. sample tube adds CD90-FITC, CD73-PE, CD14-APC 5 ul respectively, mixes; 4. sample tube adds DR-FITC, CD105-PE, CD19-APC 5 ul respectively, mixes; 5. sample tube adds CD45-FITC, CD34-PE 5 ul respectively, mixes.
(4) room temperature lucifuge is placed 15min, adds 2mL PBS/ pipe, 1400 rpm/min, centrifugal 5min.
(5) supernatant, upsprings pipe inner cell, adds 0.5mL PBS, mixes upper machine testing (if can not go up machine in time, should add paraformaldehyde to fix).
(6) upper machine analytical results obtains: CD73, CD90, CD105 are greater than 95%, positive; CD14, CD19, CD34, CD4,5HLA-DR are less than 2%, negative.
Above-mentioned explanation: the people's placenta mesenchyma stem cell obtaining according to the method separation and Culture in embodiment, meets CD73, CD90, CD105 is greater than 95%, positive; CD14, CD19, CD34, CD4,5HLA-DR are less than 2%, and negative result, through being accredited as mescenchymal stem cell.
The membrane-filter procedure using in above-mentioned aseptic testing process is as follows:
Sterility Test comprises than membrane-filter procedure and direct inoculation, and membrane-filter procedure can avoid effectively removing microbiotic and the interference of other water-soluble substanceses to aseptic detection, can reflect more accurately the state that in cell cultivation process, microorganism exists.
The aseptic detection of the present embodiment is adopted according to 2010 editions > > membrane-filter procedures of < < Chinese Pharmacopoeia and is detected, and adopts HTY-601 germ collector and APY serial culture device.The whole process of aseptic detection is all carried out in the way flow air section of 100 grades of local cleanliness factors under 10000 grades of environment cleanliness, and its whole process strictly observes aseptic technique, prevents microbial contamination.The cleanliness factor of isolated system internal medium meets the requirement of sterility test.
Concrete implementation step is as follows:
(1) take out incubator and first check that whether packing is excellent.Check and should be not more than 0.45 μ m with filter membrane aperture, diameter is about 50mm.According to trial-product characteristic, select filter membrane material, filter and filter membrane should be through suitable method sterilizings before using.During use, should guarantee the integrity of filter membrane before and after filtering.
(2) incubator is inserted one by one in stainless steel seat, pack the elastic hose of incubator into germ collector pump head, note accurate positioning, flexible pipe tendency is smooth and easy.
(3) wetting filter membrane: select sodium-chlor-peptone buffer agent of PH7.0 as washing fluid, with 75% ethanol or 0.2%, wash geramine and carefully the cleaning liquid bottle surface with plug is carried out disinfection at (position that particularly container top need to puncture), dry.Then the supravasal syringe needle of incubator is inserted in washing fluid container plug, open germ collector, adjusting rotary speed is 160RMP, after starting approximately 3 seconds, be inverted cleaning liquid bottle and be placed in and carry on bottle stand, in every cup, inject about 50ml washing fluid, wetting filter membrane (wetting liquid is without being filtered dry), takes off cleaning liquid bottle and stands in operating table surface, stops germ collector after evacuation of liquid.
(4) dilution of trial-product, transfer, filtration: with 75% ethanol or 0.2%, wash geramine and carefully sample hose dress trial-product surface is carried out disinfection, dry.Open the lid of sample hose, sample pipe shaft is held with 45 ℃ of angles, then the supravasal syringe needle of incubator is inserted to the bottom of sample hose, open germ collector, adjusting rotary speed is 160RMP, and test liquid is transferred in cup and is filtered, treat test liquid discharge to the greatest extent, stop germ collector.
(5) rinse: the elasticity helmet that takes off cup filter bowl upper port.Syringe needle is inserted in cleaning liquid bottle, open germ collector, adjusting rotary speed is 160RMP, then cleaning liquid bottle is inverted, and in every cup, injects about 100mL left and right washing fluid, rinses 3 times.After flushing completes, take off red helmet pressure release, use yellow cap stopper sealing liquid outlet (rotation push-tight), then substratum is reentered in sump pit.Pay special attention to: when rinsing, do not advise cup to carry out excessive jolting, otherwise cause washing fluid to enter air filter.
(6) add substratum:
With red intermediate plate, clamp an other pipe of incubator four-way and location buckle.With 100R rotating speed, start germ collector, then be inverted medium bottle, make all THIOGLYCOLLIC ACID salt fluid media transfer in incubator cup.First with green intermediate plate, clamp another two pipes, more red intermediate plate is taken away, inject improvement Martin substratum, equipment operation is with adding THIOGLYCOLLIC ACID salt broth.When two cups are filled it up with substratum entirely, with corresponding intermediate plate, clamp from the flexible pipe at 2～3cm place, incubator cup top, cut two flexible pipes simultaneously, leave 5～6cm, and other end is inserted to the air filter place of cup.(scissors needed spirit lamp flame)
(7) get corresponding washing fluid and operate with method, as negative control.
(8) colony culture device containing substratum of end of operation is shifted out to sterilisable chamber, get one pipe and make positive control, according to positive control bacterium selection principle, add corresponding contrast bacterium liquid.
(9) THIOGLYCOLLIC ACID salt broth is cultivated 14 days at 30～35 ℃, and improvement Martin substratum is cultivated 14 days at 23～28 ℃.Between incubation period, should check day by day whether have bacteria growing, and fill in sterility test record.As muddy or precipitation appears in trial-product pipe, in the time of can not judging from outward appearance after cultivating, desirable this nutrient solution is seeded in another identical fresh culture, microbial culture 2 days, and fungus culture 3 days, whether the fresh culture of the same race of observing inoculation occurs muddy again; Or get nutrient solution smear, and dyeing, microscopy, has judged whether bacterium.
(10) result judgement: positive control pipe is answered well-grown, and negative control pipe must not have bacteria growing.Otherwise, invalidate the test.
If trial-product pipe is all clarified, though or aobvious muddy through confirmation asepsis growth, sentence trial-product up to specification; If any aobvious muddiness confirmation have bacteria growing in trial-product pipe, sentence trial-product against regulation, unless can fully prove that experimental result is invalid, the non-trial-product of microorganism of growth is contained.When meeting following at least one condition, can sentence test-results invalid:
1. the microorganism monitored results of sterility test test equipment used and environment does not meet sterility test requirement;
2. look back sterility test process, find that there is the factor that may cause microbial contamination;
2. the microorganism growing in trial-product pipe after identifying, confirmation be article because using in sterility test and (or) aseptic technique is improper causes.If it is invalid through confirming to test, answer retry.During retry, again get with amount trial-product, check, if asepsis growth is sentenced trial-product up to specification in accordance with the law; If there is bacteria growing, sentence trial-product against regulation.
Claims (6)
1. a method for cultivator placenta mesenchyma stem cell, is characterized in that: it comprises the steps:
(1) get the human placenta of fresh collection, clean, putting into 2-8 ℃ of placenta protection liquid saves backup, described placenta protection liquid adds benzylpenicillin sodium and Vetstrep to obtain in AIM-V substratum, and wherein benzylpenicillin sodium and the Vetstrep final concentration in umbilical cord protection liquid is respectively 100-200 U/mL and 100-200 U/mL;
(2) get to be immersed in placenta protection liquid and be no more than the human placenta of 48 hours, clean, put successively again and face the sodium chloride solution that is respectively 2500-3500 U/mL and 3500-4500 U/mL with the benzylpenicillin sodium of newly joining and Vetstrep content and every milliliter and embathe containing in the Metronidazule injection of 30-50 U amphotericin B, finally clean be again placed in disposable sterilized culture dish, carry out standby;
(3) human placenta of aseptic cleaning is cut into meat gruel shape, adds AIM-V substratum, put in separator tube and carry out separation with full-automatic separate tissue device, re-use aseptic strainer filtering, obtain placenta tissue cell suspension;
(4) placenta tissue cell suspension is carried out centrifugal, abandon supernatant, collect lower floor, be placed in culturing bottle, then add wherein serum free medium, the CO that to be placed in 36-38 ℃, saturated humidity, volume fraction be 5%
2in incubator, cultivate, to observing while forming a plurality of not of uniform size, cell island that cell quantity is intensive on bottle wall, obtain people's placenta mesenchyma stem cell primary cell mixed solution, then through centrifugal, can obtain people's placenta mesenchyma stem cell primary cell, then through going down to posterity, be cultured to cytogamy degree and reach 80-90% and get final product.
2. the method for cultivator placenta mesenchyma stem cell according to claim 1, is characterized in that: the placenta protection liquid in described step (1) joins in AIM-V substratum (treatment level) again and obtains after benzylpenicillin sodium for injection and streptomycin sulphate for injection are dissolved in advance; The sodium chloride solution that the middle benzylpenicillin sodium of described step (2) and Vetstrep content are respectively 2500-3500 U/mL and 3500-4500 U/mL is mixed to get benzylpenicillin sodium for injection and streptomycin sulphate for injection by a certain amount of adding in 0.9% sodium chloride injection; Described every milliliter of preparation containing the Metronidazule injection of 30-50 U amphotericin B be by amphotericin b for inj B first with after 0.9% sodium chloride injection dissolving, then be mixed to get with a certain amount of Metronidazule injection; Described people's placenta is respectively the sodium chloride solution of 2500-3500 U/mL and 3500-4500 U/mL and every milliliter at benzylpenicillin sodium and Vetstrep content and is 8-12min containing the time of embathing in the Metronidazule injection of 30-50 U amphotericin B.
3. the method for cultivator placenta mesenchyma stem cell according to claim 1, is characterized in that: the cleaning in described step (1) and step (2) is 0.9% sodium chloride injection and cleans.
4. the method for cultivator placenta mesenchyma stem cell according to claim 1, is characterized in that: the placenta tissue in described step (1) is after getting placenta and giving birth to, and gets the umbilical cord attachment area tissue block in 3-5cm around.
5. the method for cultivator placenta mesenchyma stem cell according to claim 1, it is characterized in that: described step (4) is for cultivating after 5-6 days, not adherent upper strata is removed, then in culturing bottle, add serum free medium continuation cultivation, and changed liquid once every 3-4 days, after cultivating 10-15 days, can obtain people's placenta mesenchyma stem cell primary cell;
Then the substratum in culturing bottle is shifted out completely, toward adding mass percent in culturing bottle, it is 0.25% the tryptic phosphate buffered saline buffer submergence of recombinant animal attached cell, put into 36～38 ℃ of incubator insulation digestion 7-9 minute, when observing the contracting of attached cell circle, and after at the bottom of having departed from bottle, add the serum free medium of Digestive system equal volume to stop digestion, then be transferred in centrifuge tube, the centrifugal supernatant of abandoning, blow the even cell that is deposited in centrifuge tube bottom, then according to 4000-6000 cell/cm
2be inoculated in culturing bottle, and supplement serum free medium in culturing bottle, put 36～38 ℃, saturated humidity, the CO that volume fraction is 5%
2in incubator, cultivate, every 3-4 days full dose is changed liquid once, merge each other to attached cell, and degrees of fusion reaches 80-90%, obtains people's placenta mesenchyma stem cell, whenever necessary, can repeat aforesaid operations and carry out manyly for cell cultures, increasing.
6. the method for cultivator placenta mesenchyma stem cell according to claim 1, is characterized in that: described T25 culturing bottle can be placed people's placenta of being no more than 5g and cultivate through full-automatic separate tissue device processing, filtration, lower confluent monolayer cells after centrifugal; People's placenta that described T75 culturing bottle can be placed 10-15 g is cultivated through full-automatic separate tissue device processing, filtration, lower confluent monolayer cells after centrifugal; People's placenta that described T175 culturing bottle can be placed 30-35 g is cultivated through full-automatic separate tissue device processing, filtration, lower confluent monolayer cells after centrifugal;
The volume of the serum free medium needing in described T25 culturing bottle is 6-8mL; The volume of the serum free medium needing in described T75 culturing bottle is 20-25mL; The volume of the serum free medium needing in described T175 culturing bottle is 30-35mL.
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