CN103451151A - Method for culturing human umbilical cord mesenchymal stem cells - Google Patents
Method for culturing human umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to a method for culturing human umbilical cord mesenchymal stem cells. The method comprises the following steps: taking a freshly collected a human umbilical cord, cleaning the umbilical cord, and putting the umbilical cord into umbilical cord protection fluid to store for standby application at a temperature of 2-8 DEG C; and taking the human umbilical cord which is soaked in the umbilical cord protection fluid without exceeding 48 hours, cleaning the human umbilical cord, sequentially placing the human umbilical cord into a sodium chloride solution and a metronidazole injection to carry out immersion cleaning, cleaning the human umbilical cord and then putting the cleaned human umbilical cord into a disposable sterile petri dish for standby application, then carrying out isolated culturing until the cell fusion degree reaches 80-90%, thereby obtaining the human umbilical cord mesenchymal stem cells, wherein the sodium chloride solution is newly prepared before use, the penicillin sodium content of the sodium chloride solution is 2500-3500 U/mL, the streptomycin sulphate content of the sodium chloride solution is 3500-4500 U/mL, and the metronidazole injection contains 30-50 U of amphotericin B per milliliter. According to the method disclosed by the invention, potential hazards caused by reagents of animal serums and animal origins can be avoided; the selected human umbilical cords can be adopted for culturing the umbilical cord mesenchymal stem cells within 48 hours, therefore, the method is strong in maneuverability; the probability of unqualified mesenchymal stem cells during isolated culture caused by mycete and anaerobion pollutions can be greatly reduced.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to a kind of method of cultivator umbilical cord mesenchymal stem cells.
Background technology
Stem cell is a kind of undifferentiated cell, two fundamental characteristics are arranged, 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 generally is 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 the 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 the specific function specialization.Multipotential stem cell can be divided into the cell type contained 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, mainly be 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 the various kinds of cell type.Mescenchymal stem cell (MSC) is found in marrow, also finds subsequently to be present in many kind tissues of human body generation, growth course.In view of mescenchymal stem cell have multi-lineage potential, can hematopoiesis support and promote the characteristics 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, the 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 umbilical cord mesenchymal stem cells.
For solving technical problem of the present invention, the technical solution used in the present invention is as follows:
A kind of method of cultivator umbilical cord mesenchymal stem cells is characterized in that: it comprises the steps:
(1) get people's umbilical cord of fresh collection, clean, putting into 2-8 ℃ of umbilical cord protection liquid saves backup, described umbilical cord protection liquid adds benzylpenicillin sodium and Vetstrep to obtain in the 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 umbilical cord protection liquid and be no more than people's umbilical cord 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 embathed containing in the Metronidazule injection of 30-50 U amphotericin B, finally clean again be placed in disposable sterilized culture dish, carry out standby;
(3) people's umbilical cord of aseptic cleaning is cut into to the meat gruel shape, adds the AIM-V substratum, put in separator tube and separated with full-automatic separate tissue device, obtain umbilical cord tissue homogenate;
(4) the umbilical cord tissue homogenate of full-automatic separate tissue device being handled well is carried out centrifugal, abandons supernatant, collects lower floor, is placed in culturing bottle, then adds wherein serum free medium, the CO that to be placed in 36-38 ℃, saturated humidity, volume fraction be 5%
2in incubator, cultivate, to observing on bottle wall while forming a plurality of not of uniform size, cell island that cell quantity is intensive, obtain human umbilical cord mesenchymal stem cells primary cell mixed solution, then through centrifugal, can obtain the human umbilical cord mesenchymal stem cells primary cell, then be cultured to the cytogamy degree through going down to posterity and reach 80-90% and get final product.
Press such scheme, umbilical cord 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 the Metronidazule injection of specified quantitative; Described people's umbilical cord 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, described step (4) is for after cultivating 5-6 days, take out in not adherent another new culturing bottle of upper stratification, in former culturing bottle and new culturing bottle, all add serum free medium to continue to cultivate, and changed liquid once every 3-4 days, can obtain the human umbilical cord mesenchymal stem cells primary cell after cultivating 10-15 days;
Then the substratum in former culturing bottle and new culturing bottle is shifted out fully, toward the tryptic phosphate buffered saline buffer submergence of the recombinant animal attached cell that adds mass percent to be 0.25% in former culturing bottle and new culturing bottle, 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 broken away 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 the 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 human umbilical cord mesenchymal stem cells, whenever necessary, can repeat aforesaid operations and carry out manyly increasing for cell cultures.
Press such scheme, described T25 culturing bottle can place people's umbilical cord of being no more than 5g through full-automatic separate tissue device process, lower-hierarchy after centrifugal cultivated; People's umbilical cord that described T75 culturing bottle can be placed 10-15 g through full-automatic separate tissue device process, lower-hierarchy after centrifugal cultivated; People's umbilical cord that described T175 culturing bottle can be placed 30-35 g through full-automatic separate tissue device process, lower-hierarchy after centrifugal cultivated;
The volume of the serum free medium needed in described T25 culturing bottle is 6-8mL; The volume of the serum free medium needed in described T75 culturing bottle is 20-25mL; The volume of the serum free medium needed in described T175 culturing bottle is 30-35mL.
At first the separation preparation of human umbilical cord mesenchymal stem cells requires umbilical cord tissue to have certain activity, is generally the activity that guarantees umbilical cord tissue, need to process in time umbilical cord, and this has also brought obstacle to practical application.The present invention is placed in umbilical cord protection liquid by the umbilical cord by fresh collection and preserves, and can reach the effect to microbial disinfection residual in umbilical cord, can maintain again the purpose of the activity of umbilical cord tissue.Use 2-8 ℃ of umbilical cord of being preserved of umbilical cord protection liquid of the present invention all to can be used for the cultivation of umbilical cord mesenchymal stem cells in 48 hours, this produces to actual preparation and has brought great operability.
Except this, the present invention is when carrying out aseptic cleaning to umbilical cord, except the sodium chloride solution that uses benzylpenicillin sodium and Vetstrep carries out aseptically process, the present invention is the special self-pollution characteristics in conjunction with umbilical cord also, especially people's umbilical cord 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 human umbilical cord mesenchymal stem cells 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 purpose that it is played a role but do not affect the activity of umbilical cord mesenchymal stem cells.
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 human umbilical cord mesenchymal stem cells 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) put into umbilical cord protection liquid after the collection, can play the effect that keeps the in vitro tissue activity, make manufacture that stronger operability be arranged, separated preparation in 48 hours, still can isolate the umbilical cord mesenchymal stem cells that activity is good;
(3) the umbilical cord cleaning process is carried out aseptically process except the mycillin that uses wide spectrum, 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 caused because of mould, anerobe pollution.
Embodiment
Embodiment 1
1. ligation after delivery of baby; placenta give birth to front or give birth to after cut the about 10cm of umbilical cord; after cleaning, 0.9% sodium chloride injection joins in umbilical cord protection liquid in 2-8 ℃ of preservation; described umbilical cord 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 umbilical cord.So, umbilical cord is put in umbilical cord protection liquid and is preserved in 2-8 ℃ of preservation, can make umbilical cord all can be used for separating in 48 hours under the protection of umbilical cord protection liquid and prepare mescenchymal stem cell.
2. the aseptic cleaning of people's umbilical cord
Take out people's umbilical cord with aseptic nipper and put into sterilising vessel from umbilical cord preserving fluid, add 250mL 0.9% sodium chloride injection, with the 8cm aseptic nipper, people's umbilical cord is shaken to rinsing once back and forth.After with aseptic nipper, people's umbilical cord is transferred in another sterilising vessel again, the sodium chloride solution (dense two anti-solution) that adds 250mL to face to be respectively 3200U/mL, 4000U/mL with the benzylpenicillin sodium of newly joining and Vetstrep, every 1-2 minute, with aseptic nipper, people's umbilical cord is shaken several times back and forth, total immersion is washed 10 minutes.With aseptic nipper, people's umbilical cord is picked up again, transfer in sterilising vessel and add every milliliter of Metronidazule injection containing the 50 amphotericin b for inj B of unit, embathe 10 minutes with same method.Embathe and with the 8cm aseptic nipper, people's umbilical cord is picked up afterwards, then put in another sterilising vessel, add 0.9% sodium chloride injection, and people's umbilical cord is shaken back and forth and cleans three times, the people's umbilical cord after cleaning is placed in disposable sterilized culture dish.
Above-mentioned dense two anti-solution is for newly joining before use, and concrete compound method is: the streptomycin sulphate for injection of getting the benzylpenicillin sodium for injection of 800,000 units and 1,000,000 units respectively one 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 2.5 ten thousand units, 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 umbilical cord uses and carry out the detection of microorganism situation through membrane-filter procedure: have no bacterium, mould-growth through 14 days culture sample.
3. the separation of people's umbilical cord stem cell preparation
People's umbilical cord 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 the 5cm sterile scissors, is cut into the meat gruel shape.Get 10g at every turn and put C pipe (the full-automatic supporting separator tube of tissue processor), add the 8mLAIM-V substratum, process as stir and processed for 2 times under full-automatic tissue processor (C-01) program through full-automatic separate tissue device, then be transferred in the 50mL centrifuge tube; Use the same method and process remaining meat gruel shape human umbilical tissue, until all separated all people's umbilical cord tissue.
Full-automatic tissue processor is processed to the people's umbilical cord homogenate centrifugal 15min under 300g obtained, abandon supernatant, collect lower floor, be placed in respectively 2 T25 culturing bottles, then, after adding serum free medium wherein, 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 umbilical cord, and people's umbilical cord that the general reducible placement of T25 culturing bottle is no more than 5g is through full-automatic separate tissue device processing, tissue after centrifugal.The substratum that the T25 culturing bottle adds is 6-8mL.
4. the former culture of human umbilical cord mesenchymal stem cells
Cultivate after 5 days, get in another culturing bottle of stratification, then in two culturing bottles, all add serum free medium to proceed to cultivate, and changed liquid once every 3-4 days, after about 10-15 days, available inverted microscope has been observed the attached cell growth, then continue to cultivate, can observe attached cell and be proliferated into gradually larger cell colony.When observing the Tissue Culture Flask wall and form a plurality of not of uniform size, cell island that cell quantity is intensive, obtain human umbilical cord mesenchymal stem cells primary cell mixed solution, then, through centrifugal, can obtain the human umbilical cord mesenchymal stem cells primary cell.
5. human umbilical cord mesenchymal stem cells goes down to posterity
Substratum in culturing bottle is shifted out fully, toward the tryptic phosphate buffered saline buffer of recombinant animal that adds mass percent to be 0.25% in culturing bottle, (T25 adds 3mL, T75 adds 4mL, T175 adds 6mL), then put into 36～38 ℃ of about 7-9 minute of incubator insulation digestion, observe the contracting of attached cell circle under inverted microscope in this process, and after at the bottom of cell has broken away from bottle, add the serum free medium of Digestive system equal volume to stop digestion, then be transferred in centrifuge tube, with on binocular microscope, with tally counting and tongue thereof, expecting blue dyeing meter cytoactive.Then centrifuge tube is put to centrifugal 5min under the 300g condition, abandoned supernatant after centrifugal, with Pasteur's pipe, blow gently the even cell that is deposited in the 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 by full dose after 3-4 days and can remove not attached cell, and now cell is the spindle shape cell that degrees of fusion reaches 50% left and right.Approximately after 6 days, the cell based instinct reaches the fusion more than 80%, 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. ligation after delivery of baby; placenta give birth to front or give birth to after cut the about 10cm of umbilical cord; after cleaning, 0.9% sodium chloride injection joins in umbilical cord protection liquid in 2-8 ℃ of preservation; described umbilical cord 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 umbilical cord protection liquid is respectively 100U/mL and 100U/mL.So, umbilical cord is put in umbilical cord protection liquid and is preserved in 2-8 ℃ of preservation, can make umbilical cord all can be used for separating in 48 hours under the protection of umbilical cord protection liquid and prepare mescenchymal stem cell.
2. the aseptic cleaning of people's umbilical cord
Take out people's umbilical cord with aseptic nipper and put into sterilising vessel from umbilical cord preserving fluid, add 0.9% sodium chloride injection, with the 8cm aseptic nipper, people's umbilical cord is shaken to rinsing once back and forth.After with aseptic nipper, people's umbilical cord is transferred in another sterilising vessel again, add and face the sodium chloride solution (dense two 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 umbilical cord is shaken several times back and forth, total immersion is washed 8 minutes.With aseptic nipper, people's umbilical cord is picked up again, transfer in sterilising vessel and add every milliliter of Metronidazule injection containing the 30U amphotericin B, embathe 8 minutes with same method.Embathe and with the 8cm aseptic nipper, people's umbilical cord is picked up afterwards, then put in another sterilising vessel, add 0.9% sodium chloride injection, and people's umbilical cord is shaken back and forth and cleans three times, the people's umbilical cord after cleaning is placed in disposable sterilized culture dish.
Above-mentioned dense two 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 umbilical cord uses and carry out the detection of microorganism situation through membrane-filter procedure: have no bacterium, mould-growth through 14 days culture sample.
3. the separation of people's umbilical cord stem cell preparation
People's umbilical cord 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 the 5cm sterile scissors, is cut into the 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, process through full-automatic separate tissue device, then be transferred in the 50mL centrifuge tube; Use the same method and process remaining meat gruel shape human umbilical tissue, until all separated all people's umbilical cord tissue.
Full-automatic tissue processor is processed to the people's umbilical cord homogenate centrifugal 15min under 300g obtained, abandon supernatant, collect lower floor, be placed in culturing bottle, then, after adding serum free medium wherein, culturing bottle is put to the CO that 36～38 ℃, saturated humidity, volume fraction are 5%
2in incubator, cultivate.
4. the former culture of human umbilical cord mesenchymal stem cells
Cultivate after 5 days, get in another culturing bottle of stratification, then in two culturing bottles, all add serum free medium to proceed to cultivate, and changed liquid once every 3-4 days, after about 10-15 days, available inverted microscope has been observed the attached cell growth, then continue to cultivate, can observe attached cell and be proliferated into gradually larger cell colony.When observing the Tissue Culture Flask wall and form a plurality of not of uniform size, cell island that cell quantity is intensive, obtain human umbilical cord mesenchymal stem cells primary cell mixed solution, then, through centrifugal, can obtain the human umbilical cord mesenchymal stem cells primary cell.
5. human umbilical cord mesenchymal stem cells goes down to posterity
Substratum in culturing bottle is shifted out fully, toward the tryptic phosphate buffered saline buffer of recombinant animal that adds mass percent to be 0.25% in culturing bottle, then put into 36～38 ℃ of about 7-9 minute of incubator insulation digestion, observe the contracting of attached cell circle under inverted microscope in this process, and after at the bottom of cell has broken away from bottle, add the serum free medium of Digestive system equal volume to stop digestion, then be transferred in centrifuge tube, with on binocular microscope, with tally counting and tongue, expecting blue dyeing meter cytoactive.Then centrifuge tube is put to centrifugal 5min under the 300g condition, abandoned supernatant after centrifugal, with Pasteur's pipe, blow gently the even cell that is deposited in the centrifuge tube bottom, then according to 4000-6000 cell count/cm
2be inoculated in culturing bottle, and carry out mark, 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.After cultivating 24h, passage cell starts adherent, becomes single dispersion long strip shape cell, changes liquid by full dose after 3-4 days and can remove not attached cell, and now cell is the spindle shape cell that degrees of fusion reaches 50% left and right.Approximately after 6 days, the cell based instinct reaches the fusion more than 80%, 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 the fluidic cell detection, detection method and the results are shown in down:
Experimental principle: utilize Flow Cytometry to detect fluorescent-labeled antibody.According to the 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 known cell the intensity of entrained fluorescein, 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) the room temperature lucifuge is placed 15min, adds 2mL PBS/ pipe, 1400 rpm/min, centrifugal 5min.
(5) supernatant, will manage inner cell and upspring, and add 0.5mL PBS, mix upper machine testing (if can not go up machine in time, should add paraformaldehyde to fix).
(6) upper machine analytical results is as follows: CD73, CD90, CD105 are greater than 95%, positive; CD14, CD19, CD34, CD4,5HLA-DR are less than 2%, negative.
Above-mentioned explanation: the human umbilical cord mesenchymal stem cells obtained 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 used 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 other water-soluble substanceses to the interference of 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 " Chinese Pharmacopoeia 2010 editions " membrane-filter procedure 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 the filter membrane aperture, diameter is about 50mm.Select the filter membrane material according to the trial-product characteristic, 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 on stainless steel seat, by the elastic hose of the incubator germ collector pump head of packing into, note accurate positioning, the flexible pipe tendency is smooth and easy.
(3) wetting filter membrane: select sodium-chlor-peptone buffer agent of PH7.0 as washing fluid, wash geramine with 75% ethanol or 0.2% and carefully the cleaning liquid bottle surface with plug is carried out disinfection at (position that particularly container top need to puncture), drying.Then the supravasal syringe needle of incubator is inserted in washing fluid container plug, open germ collector, adjusting rotary speed is 160RMP, start approximately after 3 seconds, to be inverted cleaning liquid bottle and to be placed in and carry on bottle stand, inject about 50ml washing fluid in every cup, wetting filter membrane (wetting liquid is without being filtered dry), take off cleaning liquid bottle and stand on operating table surface, stops germ collector after evacuation of liquid.
(4) dilution of trial-product, transfer, filtration: wash geramine with 75% ethanol or 0.2% and carefully sample hose dress trial-product surface is carried out disinfection, drying.Open the lid of sample hose, the 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 the 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.Take off red helmet pressure release after flushing completes, 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 is carried out to excessive jolting, otherwise cause washing fluid to enter air filter.
(6) add substratum:
Clamp an other pipe of incubator four-way and location buckle with red intermediate plate.Start germ collector with the 100R rotating speed, then be inverted medium bottle, make all THIOGLYCOLLIC ACID salt fluid media transfer in the 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 fluid substratum.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, stay 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 the one pipe and make positive control, according to positive control bacterium selection principle, add corresponding contrast bacterium liquid.
(9) THIOGLYCOLLIC ACID salt fluid substratum is cultivated 14 days at 30～35 ℃, and improvement Martin substratum is cultivated 14 days at 23～28 ℃.Should check day by day whether bacteria growing is arranged between incubation period, and fill in the sterility test record.As muddy or precipitation appears in the 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 the nutrient solution smear, and dyeing, microscopy, judged whether bacterium.
(10) result judgement: the positive control pipe is answered well-grown, and the negative control pipe must not have bacteria growing.Otherwise, invalidate the test.
If the trial-product pipe is all clarified, though or aobvious muddy through the conclusive evidence asepsis growth, sentence trial-product up to specification; If in the trial-product pipe, any aobvious muddiness conclusive evidence have bacteria growing, sentence trial-product against regulation, unless energy sufficient proof 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 the sterility test requirement;
2. look back the sterility test process, find that there is the factor that may cause microbial contamination;
2. the microorganism grown in the trial-product pipe after identifying, conclusive evidence 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 the amount trial-product, check, if asepsis growth is sentenced trial-product up to specification in accordance with the law; If bacteria growing is arranged, sentence trial-product against regulation.
Claims (5)
1. the method for a cultivator umbilical cord mesenchymal stem cells, it is characterized in that: it comprises the steps:
(1) get people's umbilical cord of fresh collection, clean, putting into 2-8 ℃ of umbilical cord protection liquid saves backup, described umbilical cord protection liquid adds benzylpenicillin sodium and Vetstrep to obtain in the 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 umbilical cord protection liquid and be no more than people's umbilical cord 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 embathed containing in the Metronidazule injection of 30-50 U amphotericin B, finally clean again be placed in disposable sterilized culture dish, carry out standby;
(3) people's umbilical cord of aseptic cleaning is cut into to the meat gruel shape, adds the AIM-V substratum, put in separator tube and separated with full-automatic separate tissue device, obtain umbilical cord tissue homogenate;
(4) the umbilical cord tissue homogenate of full-automatic separate tissue device being handled well is carried out centrifugal, abandons supernatant, collects lower floor, is placed in culturing bottle, then adds wherein serum free medium, the CO that to be placed in 36-38 ℃, saturated humidity, volume fraction be 5%
2in incubator, cultivate, to observing on bottle wall while forming a plurality of not of uniform size, cell island that cell quantity is intensive, obtain human umbilical cord mesenchymal stem cells primary cell mixed solution, then through centrifugal, can obtain the human umbilical cord mesenchymal stem cells primary cell, then be cultured to the cytogamy degree through going down to posterity and reach 80-90% and get final product.
2. the method for cultivator umbilical cord mesenchymal stem cells according to claim 1 is characterized in that: the umbilical cord 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 the Metronidazule injection of specified quantitative; Described people's umbilical cord 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 umbilical cord mesenchymal stem cells 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 umbilical cord mesenchymal stem cells according to claim 1, it is characterized in that: described step (4) is for after cultivating 5-6 days, take out in not adherent another new culturing bottle of upper stratification, in former culturing bottle and new culturing bottle, all add serum free medium to continue to cultivate, and changed liquid once every 3-4 days, can obtain the human umbilical cord mesenchymal stem cells primary cell after cultivating 10-15 days;
Then the substratum in former culturing bottle and new culturing bottle is shifted out fully, toward the tryptic phosphate buffered saline buffer submergence of the recombinant animal attached cell that adds mass percent to be 0.25% in former culturing bottle and new culturing bottle, 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 broken away 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 the 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 human umbilical cord mesenchymal stem cells, whenever necessary, can repeat aforesaid operations and carry out manyly increasing for cell cultures.
5. the method for cultivator umbilical cord mesenchymal stem cells according to claim 1 is characterized in that: described T25 culturing bottle can place people's umbilical cord of being no more than 5g through full-automatic separate tissue device process, lower-hierarchy after centrifugal cultivated; People's umbilical cord that described T75 culturing bottle can be placed 10-15 g through full-automatic separate tissue device process, lower-hierarchy after centrifugal cultivated; People's umbilical cord that described T175 culturing bottle can be placed 30-35 g through full-automatic separate tissue device process, lower-hierarchy after centrifugal cultivated;
The volume of the serum free medium needed in described T25 culturing bottle is 6-8mL; The volume of the serum free medium needed in described T75 culturing bottle is 20-25mL; The volume of the serum free medium needed in described T175 culturing bottle is 30-35mL.
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| CN106719601A (en) * | 2016-11-30 | 2017-05-31 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord preserving fluid and its application |
| CN112159795A (en) * | 2020-10-13 | 2021-01-01 | 肖利霞 | Passage amplification method for umbilical cord mesenchymal stem cell working cells in public bank |
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