CN102660502B - Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell - Google Patents
Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell Download PDFInfo
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Abstract
The invention relates to methods for separating, freezing and thawing a whole cell of an umbilical cord and separating and augmenting a thawed stem cell. The method for separating and freezing the whole cell of the umbilical cord comprises the following steps of: sterilizing and cleaning an umbilical cord tissue; shearing the tissue into a block shape and carrying out digestion treatment for 1.5 hours; preparing a umbilical cord tissue frozen solution for later use; and adding the whole cell and frozen solution which are obtained by sterilizing treatment into a freezing tube, refrigerating the freezing tube for 0.5 hour at low temperature of 4DEG C, freezing the freezing tube for one day under the temperature condition of -80DEG C and then freezing the freezing tube in liquid nitrogen for later use. The method for thawing the whole cell of the umbilical cord comprises the following steps of: when the whole cell of the umbilical cord is required, extracting the whole cell of the umbilical cord from the liquid nitrogen, thawing the whole cell of the umbilical cord in a constant-temperature water bath; cleaning the whole cell of the umbilical cord by using a mesenchymal stem cell culture medium and a drop method; and augmenting a mesenchymal stem cell by using the thawed whole cell of the umbilical cord through cell culture and cell passage. According to the methods disclosed by the invention, the frozen umbilical cord tissue can be effectively protected and is convenient for thawing; and the method is especially suitable for separating and augmenting the mesenchymal stem cell after the frozen umbilical cord tissue is thawed.
Description
Technical field
The present invention relates to the method that the full cell of umbilical cord is processed, be specifically related to the full cell cryopreservation of umbilical cord, recovery to process, and by the full cellular segregation of umbilical cord after recovery and the method for expanding stem cells, be particularly related to the full cell of umbilical cord is carried out to frozen, recovery, and then the method for separated and amplification of mesenchymal stem cells therefrom.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) derives from grows early stage mesoderm and ectoderm, has the features such as multi-lineage potential, immunomodulatory and self-replacation, day by day receives people's concern.Mescenchymal stem cell is in vivo or under external specific inductive condition, can be divided into the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, after continuous passage cultivation and freezing preservation, still there is multi-lineage potential, can be used as the injuries of tissues and organs reparation that desirable seed cell causes for old and feeble and pathology, especially to treating aging and injuries of tissues and organs reparation, have very large clinical value.
MSC contains abundant in marrow, but aging along with the age, the stem cell number in marrow also can significantly reduce, the also significantly decline of proliferation and differentiation ability.In addition, marrow MSC transplants to allosome may cause immune response, and extract stem cell process to the damaging of patient and the other problems that runs into when gathering, all directly affected the clinical application of marrow MSC, making to find other alternative source for mesenchymal stem cells beyond marrow becomes an important problem.
Recent studies show that, also contains mescenchymal stem cell and energy success separation in umbilical cord tissue.This tissue-derived mescenchymal stem cell has not only kept the biological characteristics of mescenchymal stem cell, and the stem cell of separating is more original, has stronger proliferation and differentiation ability.The functionally active of its immunocyte is low, has greatly lowered the risk that triggers immune response and cause graft versus host disease (GVH disease).The infection of occult virus and microorganism and propagation probability are lower.Gatherer process is simple, to puerpera and newborn infant without any harm and damage.Above reason is enough to make umbilical cord mesenchymal stem cells to become the desirable surrogate of mesenchymal stem cells MSCs.
But umbilical cord tissue for example method and the technology of the full cellular segregation stem cell of umbilical cord is not also fully matured, and the processing of the full cell of every a umbilical cord with separated after cell cultures all need regular hour and personnel's consumption.Therefore by the full cell cryopreservation of umbilical cord and when having needs, recover and cultivate more meet cost benefit relative to the way increasing.Therefore, this area needs a full cellular segregation of simple and efficient umbilical cord, frozen, the method for recovering and increasing, to meet the demand in medicine, scientific research, the field such as clinical.
Summary of the invention
The object of the invention is to solve that prior art is frozen, the defect of the full cell method of recovery umbilical cord, the method of the full cell cryopreservation of a kind of simple and effective umbilical cord is provided, and the method for the full cell recovery of freezing and storing umbilical of the use that matches, and the method for mescenchymal stem cell amplification after recovery.The present invention finds the full cell of umbilical cord to use frozen, recovery and the separated and amplification of specific operation step; can be simply, effectively from the umbilical cord tissue primary mescenchymal stem cell of the full cellular segregation of umbilical cord for example, and can in frozen process, effectively protect primary mescenchymal stem cell.The present invention is based on this discovery and be accomplished.
Therefore, first aspect present invention provides the method for processing the full cell of umbilical cord, and the method comprises carries out separated and frozen step below to the in vitro flesh tissue of umbilical cord full cell:
(1) sterilization and cleaning: for example, with thimerosal (alcohol) umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling, for example, cleans umbilical cord tissue by damping fluid (PBS damping fluid), to reduce red corpuscle on umbilical cord tissue;
(2) digestion process: the umbilical cord tissue that step (1) is obtained is cut into tissue block, tissue block is put into digestive ferment solution (for example it comprises type i collagen enzyme, DMEM-F12 without limitation), digestion process 0.5-3 hour (for example 1.5 hours), filter and remove tissue block, add mescenchymal stem cell substratum to stop digestion, the cell then digestion being obtained carries out cell cleaning;
(3) preparation umbilical cord full cells frozen storing liquid: comprise human serum albumin and DMSO (dimethyl sulfoxide (DMSO)) in the full cells frozen storing liquid of described umbilical cord, the cold liquid storage preparing is placed on for example, under the temperature condition of 1 ℃ to 7 ℃ (4 ℃) deepfreeze;
(4) cold the depositing of the full cell of umbilical cord: for example, at 0-15 ℃ (1 ℃ to 7 ℃, for example 4 ℃) low temperature environment under, the full cells frozen storing liquid that step (3) is obtained adds in the cell after the cleaning that step (2) obtains with re-suspended cell, then re-suspended cell is added in frozen container, frozen container is put into programmed cooling device, first for example, for example, under the temperature condition of 1 ℃ to 7 ℃ (4 ℃) deepfreeze 0.2-2 hour (0.5 hour), for example, under the temperature condition of-10 ℃ to-150 ℃ (-80 ℃) freezing 0.25-3 days (for example 1 day) again, then frozen container is freezing in liquid nitrogen, standby.
According to the method for first aspect present invention, the method also comprises the following step that the full cell of the in vitro flesh tissue of frozen umbilical cord is recovered:
(5) the full cell recovery of freezing and storing umbilical: the full cell of umbilical cord that step (4) is freezing takes out from liquid nitrogen, for example thaw, to 20%-70% (50%) frozen storing liquid and start to melt, utilize mescenchymal stem cell substratum to clean cell, so that the full cell recovery of frozen umbilical cord.
According to the method for first aspect present invention, the method also comprises that the following full cell of umbilical cord to recovery carries out the step that mescenchymal stem cell is separated and increase:
(6) cell cultures: the mescenchymal stem cell substratum of adding appropriate (for example 1-6 times of cell suspension volume amount) in the cell obtaining to step (5), put into culture vessel, again culture vessel is put in incubator and cultivated, (the 3-6 days for example that is cultured to 2-7 days, for example the 4th day, for example the 5th day) time takes out culture vessel from incubator, add appropriate (for example 0.2-2 times of enchylema volume) mescenchymal stem cell substratum, continue to cultivate, for example, 8-11 days (the 9th day) times culture vessel is taken out from incubator, entirely change for the first time liquid, continue to cultivate, within every 1-3 days backward, (for example 2 days) once change liquid entirely,
(7) passage: for example, after the attached cell fusion rate in culture vessel reaches 40%-70% (60%), utilize digestive ferment (for example TrypLe Express) that attached cell is departed to container bottom, centrifugal, take supernatant liquor away, add mesenchymal stem cells substratum Eddy diffusion cell, be inoculated in that culture vessel goes down to posterity and carry out amplification cultivation, after this within every 1-3 days, (for example every 2 days) change liquid once, for example, until fusion rate reaches after 70-90% (80%), obtain umbilical cord mesenchymal stem cells, go down to posterity if desired;
And optional following one or more steps:
(8) to for step (7) gained umbilical cord mesenchymal stem cells, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(9) umbilical cord mesenchymal stem cells after step (7) gained is gone down to posterity is freezing in liquid nitrogen, standby.
According to the method for first aspect present invention, the method comprises:
(A) below, the full cell of the in vitro flesh tissue of umbilical cord is carried out to separated and frozen step:
(1) sterilization and cleaning: for example, with thimerosal (alcohol) umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling, for example, cleans umbilical cord tissue by damping fluid (PBS damping fluid), to reduce red corpuscle on umbilical cord tissue;
(2) digestion process: the umbilical cord tissue that step (1) is obtained is cut into tissue block, tissue block is put into digestive ferment solution (for example it comprises type i collagen enzyme, DMEM-F12 without limitation), digestion process 0.5-3 hour (for example 1.5 hours), filter and remove tissue block, add mescenchymal stem cell substratum to stop digestion, the cell then digestion being obtained carries out cell cleaning;
(3) preparation umbilical cord full cells frozen storing liquid: comprise human serum albumin and DMSO (dimethyl sulfoxide (DMSO)) in the full cells frozen storing liquid of described umbilical cord, the cold liquid storage preparing is placed on for example, under the temperature condition of 1 ℃ to 7 ℃ (4 ℃) deepfreeze;
(4) cold the depositing of the full cell of umbilical cord: for example, at 0-15 ℃ (1 ℃ to 7 ℃, for example 4 ℃) low temperature environment under, the full cells frozen storing liquid that step (3) is obtained adds in the cell after the cleaning that step (2) obtains with re-suspended cell, then re-suspended cell is added in frozen container, frozen container is put into programmed cooling device, first for example, for example, under the temperature condition of 1 ℃ to 7 ℃ (4 ℃) deepfreeze 0.2-2 hour (0.5 hour), for example, under the temperature condition of-10 ℃ to-150 ℃ (-80 ℃) freezing 0.25-3 days (for example 1 day) again, then frozen container is freezing in liquid nitrogen, standby,
(B) the following step that the full cell of the in vitro flesh tissue of frozen umbilical cord is recovered:
(5) the full cell recovery of freezing and storing umbilical: the full cell of umbilical cord that step (4) is freezing takes out from liquid nitrogen, for example thaw, to 20%-70% (50%) frozen storing liquid and start to melt, utilize mescenchymal stem cell substratum to clean cell, so that the full cell recovery of frozen umbilical cord;
(C) the following full cell of umbilical cord to recovery carries out the step that mescenchymal stem cell is separated and increase:
(6) cell cultures: the mescenchymal stem cell substratum of adding appropriate (for example 1-6 times of cell suspension volume amount) in the cell obtaining to step (5), put into culture vessel, again culture vessel is put in incubator and cultivated, (the 3-6 days for example that is cultured to 2-7 days, for example the 4th day, for example the 5th day) time takes out culture vessel from incubator, add appropriate (for example 0.2-2 times of enchylema volume) mescenchymal stem cell substratum, continue to cultivate, for example, 8-11 days (the 9th day) times culture vessel is taken out from incubator, entirely change for the first time liquid, continue to cultivate, within every 1-3 days backward, (for example 2 days) once change liquid entirely,
(7) passage: for example, after the attached cell fusion rate in culture vessel reaches 40%-70% (60%), utilize digestive ferment (for example TrypLe Express) that attached cell is departed to container bottom, centrifugal, take supernatant liquor away, add mesenchymal stem cells substratum Eddy diffusion cell, be inoculated in that culture vessel goes down to posterity and carry out amplification cultivation, after this within every 1-3 days, (for example every 2 days) change liquid once, for example, until fusion rate reaches after 70-90% (80%), obtain umbilical cord mesenchymal stem cells, go down to posterity if desired;
And optional
(D) following one or more step:
(8) to for step (7) gained umbilical cord mesenchymal stem cells, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(9) umbilical cord mesenchymal stem cells after step (7) gained is gone down to posterity is freezing in liquid nitrogen, standby.
According to the method for first aspect present invention, wherein said umbilical cord is the Fresh tissue of umbilical cord.
According to the method for first aspect present invention, wherein the described umbilical cord tissue of step (1) is to process in Biohazard Safety Equipment.
According to the method for first aspect present invention, wherein the step of the described tiling of step (1) is that umbilical cord tissue is laid in culture dish, the culture dish that described culture dish diameter is 5-20cm, the culture dish that preferably culture dish diameter is 10cm.
According to the method for first aspect present invention, the concentration of wherein said alcohol is 25%-95%, preferably 75%.
According to the method for first aspect present invention, the sodium salt that wherein said PBS damping fluid is phosphoric acid and/or sylvite preparation, its pH is 5.0-8.0, and preferably pH is 5.5-76, and preferably pH is 6.0-7.0.In one embodiment, in described PBS damping fluid, the concentration of phosphate radical is 0.01-0.5M, preferably 0.02-0.1M.In the present invention below tests, PBS damping fluid used is sodium phosphate salt, and wherein the concentration of phosphate radical is 0.025M, and pH is 6.5.It should be noted that, inventor's discovery, PBS buffer concentration and pH value in above-mentioned scope are little for the influential effect of the inventive method.
According to the method for first aspect present invention, wherein said digestive ferment solution is that type i collagen enzyme is added to DMEM-F12, by strainer, filtered and obtained, its digestive ferment is 0.05g-0.5g, and preferably digestive ferment is 0.08g-0.2g, preferably digestive ferment is 0.1g, its DMEM-F12 is 50-500ml, and preferably DMEM-F12 is 80-200ml, and preferably DMEM-F12 is 100ml, its strainer is 5-50 μ m strainer, preferably 20 μ m strainers.In one embodiment, described digestive ferment solution is that the type i collagen enzyme of 0.1g is joined in the DMEM-F12 of 100ml, mixes, and filtration (for example filtering with 20um strainer) obtains.
According to the method for first aspect present invention, wherein step (2) is that umbilical cord tissue is transferred in another cell cultures plate, then carry out umbilical cord tissue and be cut into tissue block, culture dish that described culture dish diameter is 5-20cm, the culture dish that preferably culture dish diameter is 10cm.
According to the method for first aspect present invention, wherein, in step (2), the size of tissue block is about 0.2-2.5 cubic centimetre, is preferably about 0.5-1.5 cubic centimetre, and preferably the cube of approximately 1 cubic centimetre is block.
According to the method for first aspect present invention, wherein in step (2), tissue block size is at 0.5-1.5 cubic centimetre, and preferred 0.5-1.0 cubic centimetre is particularly very preferred big or small approximately 1 cubic centimetre time.Although the little realization that is conducive to the inventive method of intended tissue fragment, yet the inventor finds in test under 0.2 cubic centimetre, 0.5 cubic centimetre, 1 cubic centimetre three kinds of states, they are basically identical to the digestion process effect of digestive ferment, and volume has remarkable disadvantageous effect to the digestion effect of digestive ferment after being greater than 1.5 cubic centimetres, this disadvantageous effect can weaken to a certain extent by extending digestion time.
According to the method for first aspect present invention, wherein, in step (2), the time of digestion process is 0.5-3 hour, preferably 1-2.5 hour, preferably 1.5-2 hour.It, in the time, is best to the digestion process effect of tissue block that the inventor finds the digestion process of 1-2.5 hour, both can guarantee that tissue block obtains sufficient digestion process, also can avoid cell destroyed.
According to the method for first aspect present invention, wherein, in step (2), digestion process is to carry out near temperature range body temperature, preferred 34-40 ℃, preferred 36-38 ℃, preferably 37 ℃.
According to the method for first aspect present invention, wherein, in step (2), digestion process is carried out in constant-temperature table.
According to the method for first aspect present invention, wherein, in step (2), filter removing tissue block and undertaken by filter screen, described filter screen is 50-150 μ m filter screen, preferably 100 μ m filter screens.
According to the method for first aspect present invention, wherein, in step (2), the mescenchymal stem cell substratum that stops digestion is to add according to the ratio of 2:1 ~ 1:2, the ratio of preferred 1:1, and described ratio is volume ratio.
According to the method for first aspect present invention, wherein, in step (2), the concrete steps that cell cleans are centrifugal 5-15 minute, remove supernatant liquor, add PBS damping fluid re-suspended cell, more centrifugal 5-15 minute, remove supernatant liquor.Centrifugal rotational speed is 800-2000rpm, preferred 1250rpm, and centrifugation time is preferably 10 minutes.
According to the method for first aspect present invention, in wherein said mescenchymal stem cell substratum, comprise FBS, L-Glutamine (Pidolidone), Gentamicin (gentamicin) and DMEM-F12.In one embodiment, the FBS that contains 10-20% in described mescenchymal stem cell substratum.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 15% FBS.In one embodiment, the L-Glutamine that contains 0.5-2% in described mescenchymal stem cell substratum.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 1% L-Glutamine.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 0.05% Gentamicin.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 0.05% Gentamicin.In one embodiment, the DMEM-F12 that contains 80-90% in described mescenchymal stem cell substratum.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 84% DMEM-F12.In one embodiment, the DMEM-F12 that contains the have an appointment L-Glutamine of the FBS of 15 weight parts, approximately 1 weight part, the Gentamicin of approximately 0.05 weight part and approximately 84 weight parts in described mescenchymal stem cell substratum.The inventor finds, the mescenchymal stem cell substratum of DMEM-F12 containing the have an appointment L-Glutamine of the FBS of 15 weight parts, approximately 1 weight part, the Gentamicin of approximately 0.05 weight part and approximately 84 weight parts is particularly preferred, than the arbitrary component content in making this substratum change more than 10% formula increase umbilical cord tissue adherent, shorten aspect the time equivalence fruit that attached cell climbs out of from tissue and there is significant advantage.
According to the method for first aspect present invention, in wherein said umbilical cord tissue frozen storing liquid, comprise human serum albumin and DMSO.In one embodiment, the human serum albumin that contains 55-95 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the human serum albumin that contains 70-90 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the human serum albumin that contains 80 weight parts in described umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains 4-20 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains 7-15 weight part in described umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains 10 weight parts in described umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains the have an appointment human serum albumin of 80 weight parts, approximately 10 weight parts in described umbilical cord tissue frozen storing liquid.In above-mentioned embodiment, in umbilical cord tissue frozen storing liquid, the summation of each component concentration is 100%.The inventor finds; the umbilical cord tissue frozen storing liquid (wherein the weight ratio of human serum albumin and DMSO is 80:10) of DMSO containing the have an appointment human serum albumin of 80 weight parts, approximately 10 weight parts is particularly preferred, than the formula that changes more than 10% of the arbitrary component content in making this umbilical cord tissue frozen storing liquid protecting umbilical cord tissue not to be subject to there is significant advantage aspect freezing destroyed texts.In the present invention, owing to also may containing those skilled in the art general other dosing solvent or solute in frozen storing liquid, therefore the above-mentioned human serum albumin representing with " weight part " and the amount of DMSO are a kind of relative quantities, and it can be milligram, gram, kilogram etc.
According to the method for first aspect present invention, wherein the described full cells frozen storing liquid that step (3) is obtained of step (4) adds cell after the cleaning that step (2) obtains with re-suspended cell, extracts sample and carry out cell counting after re-suspended cell.The add-on of full cells frozen storing liquid is 1-4 times of enchylema volume, for example 2 times.
According to the method for first aspect present invention, wherein the described low temperature environment of step (4) is 0-15 ℃, preferably 1 ℃ to 7 ℃, and preferably 4 ℃.
According to the method for first aspect present invention, wherein the described frozen container of step (4) is cryopreservation tube.
According to the method for first aspect present invention, the volume of the re-suspended cell frozen storing liquid wherein adding in described each the frozen container (cryopreservation tube) of step (4) is 0.5-3ml, preferably 1ml.
According to the method for first aspect present invention, wherein the density of the described cell of step (4) in frozen container be take and guaranteed that it is the upper limit that arbitrary cell all has enough DMSO that protection is provided, and described density refers to the cell individual number in unit space, and preferably 1 * 10
3-1 * 10
8every pipe, preferably 1 * 10
6every pipe.
According to the method for first aspect present invention, wherein the described programmed cooling device of step (4) is program temperature reduction box.
According to the method for first aspect present invention, wherein described deepfreeze and the cryogenic freezing of step (4) for example, put into refrigerator realization by programmed cooling device (program temperature reduction box).
According to the method for first aspect present invention, wherein the described sample refrigeration flow process of step (4) is deepfreeze 0.2-2 hour under the temperature condition of 1 ℃ to 7 ℃.In one embodiment, described deepfreeze is under the temperature condition of 2 ℃ to 6 ℃.In one embodiment, described deepfreeze is under the temperature condition of 3 ℃ to 5 ℃.In one embodiment, described deepfreeze is under the temperature condition of 5 ℃.In one embodiment, the described deepfreeze time is 0.3-1.5 hour.In one embodiment, the described deepfreeze time is 0.4-1 hour.In one embodiment, the described deepfreeze time is 0.5 hour.Inventor's discovery, under the temperature condition of 4 ℃, deepfreeze is particularly preferred in 0.5 hour, can guarantee that the full cell of DMSO and umbilical cord fully merges.Above-mentioned other refrigerating temperature conditions and cold preservation time also can make the full cytogamy of DMSO and umbilical cord, and its syncretizing effect can meet primary demand of the present invention, but under the temperature condition of 4 ℃, the deepfreeze syncretizing effect of 0.5 hour is the most abundant, has clear superiority.
According to the method for first aspect present invention, wherein the described freezing flow process of sample of step (4) is freezing 0.25-3 days under the temperature condition of-10 ℃ to-150 ℃.In one embodiment, described freezing be under the temperature condition of-30 ℃ to-120 ℃.In one embodiment, described freezing be under the temperature condition of-50 ℃ to-100 ℃.In one embodiment, described freezing be under the temperature condition of-80 ℃.In one embodiment, described freezing time is 0.4-2 days.In one embodiment, described freezing time is 0.8-1.5 days.In one embodiment, described freezing time is 1 day.The inventor finds; under the temperature condition of-80 ℃, freezing 1 day is particularly preferred, than the freezing flow process that changes more than 10% of arbitrary temperature, time in making this freezing flow process at protection umbilical cord tissue, be not subject to there is significant advantage aspect freezing destroyed texts.
According to the method for first aspect present invention, wherein described in step (5), thawing is to carry out in water bath with thermostatic control.
According to the method for first aspect present invention, in wherein said mescenchymal stem cell substratum, comprise FBS, L-Glutamine, Gentamicin and DMEM-F12.In one embodiment, the FBS that contains 10-20% in described mescenchymal stem cell substratum.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 15% FBS.In one embodiment, the L-Glutamine that contains 0.5-2% in described mescenchymal stem cell substratum.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 1% L-Glutamine (L-glutaminate).In one embodiment, the Gentamicin (gentamicin) that contains 0.02-0.1% in described mescenchymal stem cell substratum.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 0.05% Gentamicin.In one embodiment, the DMEM-F12 that contains 80-90% in described mescenchymal stem cell substratum.In one embodiment, in described mescenchymal stem cell substratum containing having an appointment 84% DMEM-F12.In one embodiment, the DMEM-F12 that contains the have an appointment L-Glutamine of the FBS of 15 weight parts, approximately 1 weight part, the Gentamicin of approximately 0.05 weight part and approximately 84 weight parts in described mescenchymal stem cell substratum.The inventor finds, the mescenchymal stem cell substratum of DMEM-F12 containing the have an appointment L-Glutamine of the FBS of 15 weight parts, approximately 1 weight part, the Gentamicin of approximately 0.05 weight part and approximately 84 weight parts is particularly preferred, than the arbitrary component content in making this substratum change more than 10% formula increase umbilical cord tissue adherent, shorten aspect the time equivalence fruit that attached cell climbs out of from tissue and there is significant advantage.
According to the method for first aspect present invention, wherein the described mescenchymal stem cell substratum of step (5) is to clean cell by topical application.Topical application can clean out in-house DMSO effectively, thereby avoids the loss of cell survival rate in recovery process.
According to the method for first aspect present invention, wherein the volume ratio of the described cell suspension of step (5) and mescenchymal stem cell substratum is 1:2-1:5, and preferred volume ratio is 1:3.
According to the method for first aspect present invention, wherein the described culture vessel of step (6) is T25 Tissue Culture Flask.
According to the method for first aspect present invention, CO in the described incubator of step (6) wherein
2concentration is 3-7%, and preferred concentration is 5%, and incubator temperature is controlled in body temperature environs, preferred 34-40 ℃, preferred 36-38 ℃, preferably 37 ℃.
According to the method for first aspect present invention, in step (8), it is to utilize trypan blue staining to count the number of frozen front and back viable cell that described cytoactive detects.
According to the method for first aspect present invention, in step (8), described cell contamination detects and utilizes a small amount of cell cultures, detects the pollution whether cell is subject to fungus and bacterium.In one embodiment, it is to utilize etiology method that described cell contamination detects, and detects cell and whether is subject to being selected from following one or more infection: Hepatitis B virus, the third liver, virus of AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.
According to the method for first aspect present invention, in step (8), it is the method for utilizing molecular genetics that described inherited disease detects, and detects freeze-stored cell and whether has inherited disease.
According to the method for first aspect present invention, in step (8), it is to detect cell HLA-ABC/DR phenotype that described HLA-ABC/DR joins type.
According to the method for first aspect present invention, in step (9), described umbilical cord mesenchymal stem cells is frozen in liquid nitrogen through programmed cooling process.
According to the method for first aspect present invention, in step (9), described umbilical cord mesenchymal stem cells is present in cells frozen storing liquid.In one embodiment, this cells frozen storing liquid comprises DMEM-F12, dimethyl sulfoxide (DMSO) and human serum albumin.In one embodiment, the DMEM-F12 that this cells frozen storing liquid comprises approximately 65 parts, the dimethyl sulfoxide (DMSO) of approximately 10 parts, the human serum albumin of approximately 15 parts.In one embodiment, this cells frozen storing liquid comprises 50% low sugar DMEM nutrient solution, 40%FBS, 10% dimethyl sulfoxide (DMSO).
According to the method for first aspect present invention, the method comprises the following steps:
(1) sterilization and cleaning: in Biohazard Safety Equipment, with alcohol, umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from centre, be laid on aseptic 10cm cell cultures plate, utilize PBS cleansing tissue, to reduce to organize red corpuscle above;
(2) digestion process: the DMEM-F12 that the type i collagen enzyme of 0.1g is added to 100ml, then with 20 μ m strainers, filter and obtain Digestive system, the umbilical cord tissue that step (1) is obtained is transferred in another 10cm cell cultures plate, and umbilical cord tissue is cut into 1cm
3the tissue block of size, tissue block is put in the Digestive system having prepared, in the constant-temperature table of 37 ℃, digest 1.5 hours, utilize 100 μ m filter screens to remove remaining tissue block, by the volume ratio of 1:1, add mescenchymal stem cell substratum (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to stop digestion, then with rotating speed 1250rpm centrifugal 10 minutes, supernatant liquor is removed and add after PBS re-suspended cell again with rotating speed 1250rpm centrifugal 10 minutes;
(3) preparation umbilical cord tissue frozen storing liquid: the DMSO (dimethyl sulfoxide (DMSO)) of the human serum albumin that comprises 80 weight parts in described umbilical cord tissue frozen storing liquid and 10 weight parts, the cold liquid storage preparing is placed on 4 ℃ of Refrigerator stores until use;
(4) cold the depositing of the full cell of umbilical cord: the frozen storing liquid that 2ml step (3) is obtained is added in the cell after the cleaning that step (2) obtains with re-suspended cell, extract sample in a small amount and carry out cell counting, then re-suspended cell is managed 1ml with each, cell density 1 * 10
6every pipe adds in cryopreservation tube, and this process need be carried out under the cold condition of 4 ℃, and cryopreservation tube is put in program temperature reduction box, first deepfreeze 0.5 hour under the temperature condition of 4 ℃, under the temperature condition of again-80 ℃ freezing 1 day, then cryopreservation tube is freezing in liquid nitrogen, standby.
Further, can comprise and the matching used method for resuscitation of cryopreservation methods:
(5) the full cell recovery of freezing and storing umbilical: the full cell of umbilical cord that step (4) is freezing takes out from liquid nitrogen, be placed in water bath with thermostatic control and thaw and start to melt to half frozen storing liquid, utilize mescenchymal stem cell substratum (it for example comprises 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to carry out topical application and clean the full cell of umbilical cord.
Further, can comprise the method that the rear mescenchymal stem cell of recovery is separated and increase:
(6) cell cultures: the cell 1ml that step (5) is obtained adds the mescenchymal stem cell substratum of 6ml, transfers to T25 culturing bottle, then puts T25 culturing bottle into CO
2concentration is to cultivate in 37 ℃ of incubators of 5%, while being cultured to the 5th day, T25 culturing bottle is taken out from incubator, add 3ml mescenchymal stem cell substratum, continue to cultivate, in the time of the 9th day, T25 culturing bottle is taken out from incubator, entirely change for the first time liquid, continue to cultivate, within every 2 days backward, once entirely change liquid;
(7) passage: the attached cell fusion rate when T25 culturing bottle the inside reaches 80% left and right, can utilize digestive ferment (TrypLE Express) that attached cell is departed to T25 culturing bottle bottom, after centrifugal, remove supernatant liquor, and add mescenchymal stem cell substratum Eddy diffusion cell, be inoculated in T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this within every two days, change liquid once until after fusion rate reaches 80%, obtain, go down to posterity again if desired.
Further, can, for above step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type.
Further, the umbilical cord mesenchymal stem cells after above step (8) gained can being gone down to posterity is frozen in liquid nitrogen, standby.
Further, can be by the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and the biological characteristics and the multi-lineage potential that carry out cell are identified, and cell is carried out to molecular genetics diagnosis, preserve all related datas of cell, set up the database of umbilical cord stem cell and carry out associated with freeze-stored cell.Therefore in the present invention in one aspect, the method of setting up umbilical cord stem cell database is provided, it comprises that first aspect present invention is separated and the step of the umbilical cord mesenchymal stem cells that increases, and following steps: by the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and the biological characteristics and the multi-lineage potential that carry out cell are identified, and cell is carried out to molecular genetics diagnosis, preserve all related datas of cell, set up the database of umbilical cord stem cell and carry out associated with freeze-stored cell.
In addition,, in a first aspect of the present invention, provide the method for and amplification separated to stem cell after the full cell cryopreservation of umbilical cord, recovery and recovery.Therefore second aspect present invention provides a kind of umbilical cord mesenchymal stem cells.
According to the umbilical cord mesenchymal stem cells of second aspect present invention, it obtains according to method described in the arbitrary embodiment of first aspect present invention.
According to the umbilical cord mesenchymal stem cells of second aspect present invention, its cell purity is greater than 90%, for example, be greater than 95%.In one embodiment, described umbilical cord mesenchymal stem cells is after in 3 generations, went down to posterity above, and cell purity is greater than 90%, for example, be greater than 95%.
Below the present invention is further illustrated.The document that the present invention quotes, and the document of quoting in the document, their full content is incorporated to herein by reference.
In the present invention, in arbitrary technical scheme of either side of the present invention, its arbitrary technical characterictic is equally applicable to arbitrary embodiment of either side of the present invention, as long as they can not cause contradiction, and this being mutually useful in if desired can be done suitable modification.
In the present invention, term " umbilical cord mesenchymal stem cells " refers to the mescenchymal stem cell that derives from umbilical cord.Therefore in the present invention, particularly relate in linguistic context of the present invention, term " umbilical cord mesenchymal stem cells " can exchange and use with " umbilical cord stem cell ", " stem cell ", " mescenchymal stem cell ", unless separately had clearly and indicated.
In the present invention, term " PBS damping fluid " or " PBS " refer to phosphate buffered saline buffer.Those skilled in the art know the generality formula of the PBS using under situation of the present invention and compound method and their general aspects for example pH value or pH scope.
In the present invention, term " umbilical cord " refers to neonatal umbilical cord, refers to especially the umbilical cord within 4 hours postpartum.
Mescenchymal stem cell (mesenchymal stem cell, MSC) for example the mankind's mescenchymal stem cell is separated the earliest from marrow, derive from the tissue stem cell that a mesoblastic class has multi-lineage potential and self-renewal capacity, have to scleroblast in vivo with under external specified conditions, chondrocyte, adipocyte, endotheliocyte, neurocyte, myocyte, the ability of the multiple adult cytodifferentiation such as liver cell (Caplan AI.Mesenchymal stem cells.J Orthop Res.1991, 9:641-650.Pittenger MF, Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999, 284:143-147).Up-to-date research shows that mescenchymal stem cell has immunomodulatory and Hematopoiesis Support affect, and is easy to foreign gene importing expression.Therefore mescenchymal stem cell tissue-engineered bone, cartilage and the cardiac muscle seed cell in building still not, important carrier cell in gene therapy, and because mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant, in hematopoietic stem cell transplantation and organ transplantation, be with a wide range of applications.Mescenchymal stem cell has the characteristic of external adherent growth, utilizes this specific character, and people success separation and Culture from the Various Tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood go out mescenchymal stem cell.
The mescenchymal stem cell of reporting is at present mainly derived from marrow, adopts density gradient centrifugation to obtain.Although separation method is easy, donor is got marrow need to experience a more painful operation, and in the process of drawing materials and after drawing materials, has very high infection chance; Because the content of MSC in human bone marrow is extremely rare, every 10
5～10
6in individual mononuclearcell, approximately only have 1, and along with the increase at age, in marrow, the quantity of mescenchymal stem cell, propagation and differentiation capability all significantly decline, it is restricted in research with in applying especially clinical application.The umbilical cord that originates from embryonic development period extraembryonic mesoderm is comprised of interstitial, blood vessel and nurse cell, contains a large amount of mesenchyme compositions.
Up-to-date research shows to contain abundant stem cell in umbilical cord, and from umbilical cord, separation and Culture goes out these multipotential stem cells and will open up a brand-new and abundant source for experimental study and clinical application.
Thereby existing separate stem cells is set up the method for stem cell bank, still have shortcomings, for example purity is not enough and/or quantity is not high, and then demonstrates these methods and still can not meet people's expectation.The for example invention of CN 101270349A (Chinese Patent Application No. 200810061267.6, open day on September 24th, 2008) disclosed being entitled as " placenta mesenchyma stem cell separation and amplification in vitro cultural method "; The invention of CN 101693884A (Chinese Patent Application No. 200910117522.9, open day on April 14th, 2010) disclosed being entitled as method of separating and extracting stem cells " a kind of from placenta, umbilical cord or fatty tissue "; The invention that CN 102146359A (Chinese Patent Application No. 201110005964.1, open day on August 10th, 2011) disclosed being entitled as " extracted the method for primary mesenchymal stem cells and serum-free amplification " from placenta.These methods are remaining to be further improved aspect the purity of extract and/or the rate of recovery.
Owing to containing abundant hemopoietic stem cell in bleeding of the umbilicus, people set up unbilical blood bank these important Biological resources of umbilical hemopoietic stem cell are stored, for multiple disease in the blood system and disease of immune system provide a kind for the treatment of means.Same umbilical cord mesenchymal stem cells is as a kind of more importantly stem cell resource, we use conventional cell freezing method to be chilled in the medium-term and long-term preservation of profound hypothermia liquid nitrogen of-196 degrees Celsius, set up umbilical cord stem cell bank, for stem-cell therapy is in the future preserved seed.
The method according to this invention, the freezing flow process of the formula of umbilical cord tissue frozen storing liquid and sample can successfully and effectively be protected in freezing process the full cell of umbilical cord.The method according to this invention, utilize topical application substratum to be added to the full cell of freezing and storing umbilical of recovery, not only the DMSO in full cell can be cleaned out, can also avoid using centrifugal step, thereby avoid the loss of cell in centrifugal process, and then reach the effect of the survival rate of effective raising recovery cell.The method according to this invention, wherein mescenchymal stem cell culture medium prescription can successfully and effectively carry out amplification in vitro to umbilical cord mesenchymal stem cells.The method according to this invention, the setting of wherein changing the liquid time has been shortened attached cell and has been reached the time of specifying fusion rate.
The present invention is simple to operate; convenient and practical; can effectively protect the full cell of frozen umbilical cord; avoid cell survival rate in recovery process to run off; can obtain a large amount of mescenchymal stem cells; differentiation performance is good, has to the ability of the cytodifferentiation such as scleroblast, adipocyte, chondrocyte, endotheliocyte, neurocyte.With now methodical comparison: at present MSC mainly adopts modus operandi to extract donor marrow or the separated umbilical cord of perfusion method, adherent culture acquisition.It is few that this method is got cell quantity, and donor is being got the possibility that all has infection after marrow is got in marrow neutralization.The present invention's success is separated in umbilical cord obtains the higher mescenchymal stem cell of a large amount of purity, and uses this method to set up umbilical cord stem cell bank and lay in this stem cell that has application prospect.This method is simple and easy to do, and because umbilical cord is the same with bleeding of the umbilicus, Cell Component is inmatureer, and wide material sources are conveniently easy to get, and therefore method of the present invention will have prospect widely in the clinical application of stem cell.
Embodiment
By the following examples, can conduct further description the present invention, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and is not deviating under the prerequisite of the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the test method that use in test.Although be well known in the art for realizing many materials and the working method that the object of the invention used, the present invention still does detailed as far as possible description at this.
the side of stem cell separation and amplification after embodiment 1, the full cell cryopreservation of umbilical cord, recovery and recovery
method
Umbilical cord tissue cryopreservation methods comprises the following steps:
(1) sterilization and cleaning: in Biohazard Safety Equipment, with alcohol, umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from centre, be laid on aseptic 10cm cell cultures plate, utilize PBS cleansing tissue, to reduce to organize red corpuscle above;
(2) digestion process: the DMEM-F12 that the type i collagen enzyme of 0.1g is added to 100ml, then with 20 μ m strainers, filter and obtain Digestive system, the umbilical cord tissue that step (1) is obtained is transferred in another 10cm cell cultures plate, and umbilical cord tissue is cut into 1cm
3the tissue block of size, tissue block is put in the Digestive system having prepared, in the constant-temperature table of 37 ℃, digest 1.5 hours, utilize 100 μ m filter screens to remove remaining tissue block, by the volume ratio of 1:1, add mescenchymal stem cell substratum (wherein comprising 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to stop digestion, then with rotating speed 1250rpm centrifugal 10 minutes, supernatant liquor is removed and add after PBS re-suspended cell again with rotating speed 1250rpm centrifugal 10 minutes;
(3) preparation umbilical cord tissue frozen storing liquid: the DMSO (dimethyl sulfoxide (DMSO)) of the human serum albumin that comprises 80 weight parts in described umbilical cord tissue frozen storing liquid and 10 weight parts, the cold liquid storage preparing is placed on 4 ℃ of Refrigerator stores until use;
(4) cold the depositing of the full cell of umbilical cord: the frozen storing liquid that 2ml step (3) is obtained is added in the cell after the cleaning that step (2) obtains with re-suspended cell, extract sample in a small amount and carry out cell counting, then re-suspended cell is managed 1ml with each, cell density 1 * 10
6every pipe adds in cryopreservation tube, and this process need be carried out under the cold condition of 4 ℃, and cryopreservation tube is put in program temperature reduction box, first deepfreeze 0.5 hour under the temperature condition of 4 ℃, under the temperature condition of again-80 ℃ freezing 1 day, then cryopreservation tube is freezing in liquid nitrogen, standby.
Comprise the following steps with the matching used method for resuscitation of cryopreservation methods:
(5) the full cell recovery of freezing and storing umbilical: the full cell of umbilical cord that step (4) is freezing takes out from liquid nitrogen, be placed in water bath with thermostatic control and thaw and start to melt to half frozen storing liquid, utilize mescenchymal stem cell substratum (wherein comprising 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to carry out topical application and clean the full cell of umbilical cord.
After recovery, mescenchymal stem cell method separated and amplification comprises the following steps:
(6) cell cultures: the cell suspension 6ml that step (5) is obtained adds the mescenchymal stem cell substratum of 6ml, transfers to T25 culturing bottle, then puts T25 culturing bottle into CO
2concentration is to cultivate in 37 ℃ of incubators of 5%, while being cultured to the 5th day, T25 culturing bottle is taken out from incubator, add 3ml mescenchymal stem cell substratum, continue to cultivate, in the time of the 9th day, T25 culturing bottle is taken out from incubator, entirely change for the first time liquid, continue to cultivate, within every 2 days backward, once entirely change liquid;
(7) passage: the attached cell fusion rate when T25 culturing bottle the inside reaches 80% left and right, can utilize digestive ferment (TrypLE Express) that attached cell is departed to T25 culturing bottle bottom, after centrifugal, remove supernatant liquor, and add mescenchymal stem cell substratum Eddy diffusion cell, be inoculated in T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this within every two days, change liquid once until fusion rate goes down to posterity after reaching 80% again.
In the test of above step, the full cell of umbilical cord after recovery occurs the attached cell that starts to have for the 13rd day of cultivating, and be cultured to the 23rd day cell confluency and reach 80%, after 3 generations went down to posterity, cell purity is greater than 90%.
In the test of above step; the DMSO of the human serum albumin that comprises 80 weight parts in umbilical cord tissue frozen storing liquid and 10 weight parts (dimethyl sulfoxide (DMSO)); in two kinds of components of this frozen storing liquid; any one ratio is departing from said ratio 20% when above; frozen storing liquid can not effectively be protected umbilical cord tissue in freezing process, is embodied in the rear separated mescenchymal stem cell counting of freezing and storing umbilical tissue recovery and obviously declines.
In the test of above step; the freezing flow process of sample is deepfreeze 0.5 hour under the temperature condition of 4 ℃; under the temperature condition of again-80 ℃ freezing 1 day; the arbitrary temp of this freezing flow process is departing from said temperature 60% when above; and the time departing from 80% when above; in refrigerating process, umbilical cord tissue is not effectively protected, and is embodied in the rear separated mescenchymal stem cell counting of freezing and storing umbilical tissue recovery and obviously declines.
In the test of above step, in mescenchymal stem cell substratum, comprise 15 weight part FBS, 1 weight part L-Glutamine, 0.05 weight part Gentamicin and 84 weight part DMEM-F12.In four kinds of components of this substratum, any three kinds of ratios are wherein fixedly time, and alternative ratio, departing from said ratio 10% when above, is passaged to after T25 culturing bottle, the 17th day fusion rate, does not all reach 75%.For example in the mescenchymal stem cell substratum using, comprise 1 weight part L-Glutamine, 0.05 weight part Gentamicin and 84 weight part DMEM-F12, and when 12 weight part FBS, 13.5 weight part FBS, 16.5 weight part FBS or 18 weight part FBS, in four kinds of situations, be passaged to after T25 culturing bottle, the 17th day fusion rate all between 53-74%.
In the test of above step, changing the liquid time is the liquid that entirely changes for the first time for the 9th day of cell cultures, and backward within every 2 days, once entirely changing liquid, the liquid time of changing arbitrarily is wherein being departed from the above-mentioned time 10% when above, and attached cell did not reach fusion rate 80% in 20 days.That for example changes the liquid time and be cell cultures changes liquid on the 12nd day for the first time entirely, or backward within every 4 days, once entirely to change liquid, in two kinds of situations, attached cell reached 80% at 22-25 days
In the test of above step, Digestive system is for adding the type i collagen enzyme of 0.1g the DMEM-F12 of 100ml, and digestion time is 1.5 hours.In nutrient solution, type i collagen enzyme content departs from above-mentioned content 10% when above, or departs from the above-mentioned time 10% when above in digestion time, and whether the full cell in tissue can not high efficiency separation, separated efficiently according to extracting the cell counting judgement of sample in a small amount.
the side of stem cell separation and amplification after embodiment 2, the full cell cryopreservation of umbilical cord, recovery and recovery
method
The method of reference example 1 is carried out.The full cell of umbilical cord after recovery occurs the attached cell that starts to have for the 14th day of cultivating, is cultured to the 24th day cell confluency and reaches 80%.After 3 generations went down to posterity, cell purity is greater than 90%.
the side of stem cell separation and amplification after embodiment 3, the full cell cryopreservation of umbilical cord, recovery and recovery
method
The method of reference example 1 is carried out.The full cell of umbilical cord after recovery occurs the attached cell that starts to have for the 13rd day of cultivating, is cultured to the 24th day cell confluency and reaches 80%.After 3 generations went down to posterity, cell purity is greater than 85%.
the side of stem cell separation and amplification after embodiment 4, the full cell cryopreservation of umbilical cord, recovery and recovery
method
The method of reference example 1 is carried out.The full cell of umbilical cord after recovery occurs the attached cell that starts to have for the 12nd day of cultivating, is cultured to the 23rd day cell confluency and reaches 80%.
the side of stem cell separation and amplification after embodiment 5, the full cell cryopreservation of umbilical cord, recovery and recovery
method
The method of reference example 1 is carried out.The full cell of umbilical cord after recovery occurs the attached cell that starts to have for the 14th day of cultivating, is cultured to the 23rd day cell confluency and reaches 80%.
the cultivation and frozen of going down to posterity of embodiment 6, umbilical cord MSC
The cell that embodiment 1-5 any one is obtained digests, and gets 1 * 10 after digestion
6cell joins in 1ml cells frozen storing liquid (containing 65 parts of DMEM-F12+10 part dimethyl sulfoxide (DMSO)+15 part human serum albumins), finally enters into liquid nitrogen container frozen through programmed cooling.
the Identification of Biological Characteristics of embodiment 7, umbilical cord MSC
1, Growth of Cells and Morphological Characteristics thereof
By the separation and Culture of embodiment 1 and embodiment 6, umbilical cord mononuclearcell was cultivated after 72 hours can obviously see fusiformis attached cell under the microscope, about 10 days, can form turbine-like cell clone, can form the adherent layer that merge 80% left and right after had digestive transfer culture.In culturing process, find the relative homogeneous of this cellular form, rate of propagation is fast, and adherent speed is fast, easily by trysinization, is passaged to 5-15 generation, and its form and growth characteristic are also without obviously changing.
2, flow cytometry identification of M SC surface marker
Get respectively the 3rd, 6,9,12,15 generation cell, Flow cytometry cell surface marker, dynamically observes the variation of cell surface marker in culturing process.Digestion collecting cell, gets 8 * 10 after counting
6individual cell, packing 16 pipes; PBS washes once, the centrifugal 10min of 1500rpm; Abandon supernatant, residual 100～200 μ l, piping and druming mixes cell; Add CD45, CD105, HLA-ABC, HLA-DR, each 10 μ l of UEA-1 antibody of CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibody and the FITC mark of PE mark, and to establish a pipe be blank; At 4 ℃, lucifuge reaction 30min; PBS washes once, the centrifugal 10min of 1500rpm; Directly the cell of mark is abandoned supernatant, adds 200 μ l PBS piping and druming to mix cell, and 1% paraformaldehyde of 200 μ l is fixed, put 4 ℃ to be measured, upflowing cell instrument detects in 3 days.
Flow cytometer detects the surface marker of cell, dynamically observes the cell in the 3rd, 6,9,12,15 generations, without obviously changing.Not expressing hematopoietic cell surface marker is CD14, CD31, CD34 (HSPC and endotheliocyte are positive), CD45 (white corpuscle is positive), CD54 (ICAM-1), CD80 (B7-1), the lasting feminine gender of CD86 (B7-2), HLA-DR (MHC-II quasi-molecule), CD29 and CD44 (acceptor of scleroproein and transparency grease hydrochlorate, stroma cell is expressed), CD73 (being SH-3,4), CD105 (being SH-2), CD166 (mesenchymal cell expression), HLA-ABC (MHC-I quasi-molecule) and UEA-1 (surface marker of endotheliocyte) are continuously the positive.After in 3 generations, went down to posterity above, cellular constituent homogeneous, purity is more than 95%.
3, the cell cycle of Flow cytometry umbilical cord MSC
When cell grows to 80% left and right and merges, digestion collecting cell approximately 1 * 10
6individual, PBS washes once, adds 70% ethanol to fix, and 4 ℃ to be measured.During detection, the first centrifugal ethanol that goes, then wash once with PBS, add RNase I 500u, 37 ℃ of reaction 30min, PBS washes once, adds propidium iodide (PI, final concentration 50 μ g/ml) 1ml, room temperature lucifuge reaction 20min, upper machine testing cell DNA content.
After measured the 3rd generation and the 6th generation cell DNA content, cell cycle analysis, G
0/ G
1phase, S phase and G
2m phase proportion is respectively 96.35%, 96.66%, 1.11%, 0.09%, and 2.54%, 3.25%.Result shows that the cell of vitro culture has typical stem cells hyperplasia feature, only has a few cell in active proliferation period (1.11%, 0.09%), and most cell is in quiescent stage (96.35%, 96.66%).
4, the drafting of umbilical cord MSC growth curve and the mensuration of logarithmic phase doubling time
The cell in vegetative period of taking the logarithm, digestion counting, makes cell suspension (2 * 104/ml) with the LG-DMEM substratum of 10%FBS, every hole inoculation 0.5ml in 24 orifice plates, 37 ℃, 5%CO
2, under saturated humidity, cultivate.Get 3 multiple holes every day, living cell counting number after Trypan Blue, calculating mean value, Continuous Observation 7 days.Take incubation time as transverse axis, and cell count is the longitudinal axis, draws cell growth curve.With Patterson formula, calculate cell in the doubling time of logarithmic phase, i.e. Td=Tlg2/Lg (Nt/No), Td: doubling time (h), T: cell increases to Nt time used (h), N: cell count by No.
By Cytometric result drafting every day cell growth curve, calculate the doubling time.By cell growth curve, can be found out, cell at 2-4 days in exponential phase of growth.According to formula calculate the 5th generation cell in doubling time of exponential phase of growth within the scope of 18-30 hour.
5, the evaluation of umbilical cord MSC multi-lineage potential
(1) osteogenic induction
3 above MSC of generation, by 1 * 10
5six orifice plates are inoculated in/hole, are put in 37 ℃, 5%CO
2, under saturated humidity, in MSC substratum, cultivate after 24h, use instead containing 10% DMEM-HG through screening FBS and add dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM, be put in 37 ℃, 5%CO
2, under saturated humidity, cultivate, within every 3 days, half amount is changed liquid, coinduction 2-4 week.Alkaline phosphatase staining identifies that scleroblast forms, and Von Kossa dyeing identifies that bone tubercle forms.
Containing 10% DMEM-HG through screening FBS, add dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM to cultivate 1 week, cellular form occurs significantly to change, from fusiform inoblast sample, become polygon, be similar to neuronal cell sample, cell periphery occurs that long filament shape is outstanding, and can extend towards periphery.Continue to cultivate 2 weeks above after, in cell matrix, there is calcified plaque, mineralizer engenders, and starts to form the little junction structure of multilayer, to cultivating after 4 weeks, visible obvious calcification tubercle.In the time of 2 weeks, alkaline phosphatase staining is strong positive reaction, reaches more than 95%, and the control group of not induced is most of negative, only less than 5%, is shown as the weak positive, shows that cell transforms to scleroblast.Von Kossa dyeing can be dyed black by the calcium depositing in bone tubercle, and the visible a large amount of black bone tubercle of induction group, have obvious three-dimensional arrangement, and control group does not all have positive reaction at any time.
(2) Adipogenic induction
3 above MSC of generation, by 1 * 10
5/ hole is inoculated in six orifice plates, is put in 37 ℃, 5%CO
2, under saturated humidity, in MSC substratum, cultivate after 24h, use instead containing 10% DMEM in high glucose through screening FBS, and add dexamethasone 1 μ M, INDOMETHACIN 60 μ M, IBMX 0.5mM, Regular Insulin 5 μ g/ml, be put in 37 ℃, 5%CO
2, under saturated humidity, cultivate, within every 3 days, half amount is changed liquid, coinduction 2 weeks, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS, add dexamethasone 1 μ M, INDOMETHACIN 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml to cultivate 3 days, there is form and change in cell, by fusiform inoblast sample, is shunk and shorten gradually, and 90% above cell becomes cube or polygon; Cultured continuously 7 days, has small fat to ooze existing in visible cell under mirror, along with the prolongation of incubation time, fat drips and increases gradually and merge, and when cultivating 2 weeks, merges as seen agglomerating fat and drips and be full of whole cell.The fat producing in oil red O stain visible cell is dyed redness by specificity.
(3) become chondrocyte induction
3 generation above cell, according to every pipe 2 * 10
5cell divides and installs to 15ml polypropylene centrifuge tube, low-speed centrifugal makes cell in test tube, form micelle, in containing the DMEM-HG of 2.5%FBS, add Regular Insulin, Transferrins,iron complexes, each 6.25 μ g/ml of Sodium Selenite, BSA 1.25 μ g/ml, Sodium.alpha.-ketopropionate 1mM/L, xitix phosphoric acid 37.5 μ g/ml, TGF-β
150ng/ml, is put in 37 ℃, 5%CO
2, cultivate under saturated humidity, within every 3 days, half amount is changed liquid, cultured continuously 2 weeks.
Induce after 2 weeks cell micelle is broken up to smear, dye visible II Collagen Type VI of alcian blue (Alcian blue) forms extracellular matrix and is blue, and control group dyes without indigo plant.
By the detection of above a series of data targets, demonstrate the MSC that the separation of application the inventive method obtains, have to the ability of scleroblast, adipocyte, Chondrocyte Differentiation, the MSC that proved inventive method obtains has stem cell characteristic.
the foundation of embodiment 8, umbilical cord stem cell bank
1, the detection of cytoactive
Utilize trypan blue staining to count the number of frozen front and back viable cell.
2, the detection of cell contamination
Utilize a small amount of cell cultures, detect the pollution whether cell is subject to fungus and bacterium.Utilize etiology method, detect whether cell is subject to Hepatitis B virus, the third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST infects.
3, the detection of inherited disease
Utilize the method for molecular genetics, detect freeze-stored cell and whether have inherited disease.
4, HLA-ABC/DR joins type
Detect cell HLA-ABC/DR phenotype, and place on record.
5, the investigation of cell derived
Record fetus and father and mother's thereof detail file, and place on record.
6, the foundation of umbilical cord stem cell database
After preserving normal umbilical cord stem cell, set up the database of umbilical cord stem cell, comprising first five items data, and foundation and freeze-stored cell is associated.
Claims (4)
1. process the method for the full cell of umbilical cord, the method comprises:
(A) below, the full cell of the in vitro flesh tissue of umbilical cord is carried out to separated and frozen step:
(1) sterilization and cleaning: with thimerosal alcohol, umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling, by PBS buffer solution for cleaning umbilical cord tissue, to reduce the red corpuscle on umbilical cord tissue;
(2) digestion process: the umbilical cord tissue that step (1) is obtained is cut into tissue block, tissue block is put into digestive ferment solution, and digestion process 1.5 hours, filters and removes tissue block, add mescenchymal stem cell substratum to stop digestion, the cell then digestion being obtained carries out cell cleaning;
(3) preparation umbilical cord full cells frozen storing liquid: the human serum albumin that comprises 80 weight parts in the full cells frozen storing liquid of described umbilical cord and the DMSO of 10 weight parts, the frozen storing liquid preparing is placed on deepfreeze under the temperature condition of 1 ℃ to 7 ℃;
(4) the full cell cryopreservation of umbilical cord: under the low temperature environment of 1 ℃ to 7 ℃, the full cells frozen storing liquid that step (3) is obtained joins in the cell after the cleaning that step (2) obtains with re-suspended cell, then re-suspended cell is added in frozen container, frozen container is put into programmed cooling device, first deepfreeze 0.5 hour under the temperature condition of 4 ℃, under the temperature condition of-80 ℃ freezing 1 day again, then frozen container is freezing in liquid nitrogen, standby;
(B) the following step that the full cell of the in vitro flesh tissue of frozen umbilical cord is recovered:
(5) the full cell recovery of freezing and storing umbilical: the full cell of umbilical cord that step (4) is freezing takes out from liquid nitrogen, thaws and starts to melt to 20%-70% frozen storing liquid, utilizes mescenchymal stem cell substratum to clean cell, so that the full cell recovery of frozen umbilical cord;
(C) the following full cell of umbilical cord to recovery carries out the step that mescenchymal stem cell is separated and increase:
(6) cell cultures: the mescenchymal stem cell substratum of adding 1-6 times of cell suspension volume amount in the cell obtaining to step (5), put into culture vessel, again culture vessel is put in incubator and cultivated, while being cultured to 2-7 days, culture vessel is taken out from incubator, add the mescenchymal stem cell substratum of 0.2-2 times of enchylema volume, continue to cultivate, in the time of the 9th day, culture vessel is taken out from incubator, entirely change for the first time liquid, continue to cultivate, within every 2 days backward, once entirely change liquid;
(7) passage: the attached cell fusion rate in culture vessel reaches 40%-70%, utilize digestive ferment TrypLeExpress that attached cell is departed to container bottom, centrifugal, take supernatant liquor away, add mescenchymal stem cell substratum Eddy diffusion cell, be inoculated in that culture vessel goes down to posterity and carry out amplification cultivation, after this every 1-3 days changes liquid once, until fusion rate reaches after 70-90%, obtain umbilical cord mesenchymal stem cells, go down to posterity if desired;
And
(D) the following step:
(8), to step (7) gained umbilical cord mesenchymal stem cells, detect at least one item of following items: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(9) umbilical cord mesenchymal stem cells after step (7) gained is gone down to posterity is freezing in liquid nitrogen, standby,
Wherein,
Digestive ferment solution described in step (2) is that the type i collagen enzyme of 0.1g is joined in the DMEM-F12 of 100ml, mixes, and filtration obtains,
The FBS that contains 15 weight parts in described mescenchymal stem cell substratum, the L-Glutamine of 1 weight part, the Gentamicin of 0.05 weight part and the DMEM-F12 of 84 weight parts.
2. according to the process of claim 1 wherein in step (5), what utilize the full cell employing of mescenchymal stem cell substratum cleaning umbilical cord is topical application.
3. according to the process of claim 1 wherein that described PBS damping fluid is sodium phosphate salt, wherein the concentration of phosphate radical is 0.025M, and pH is 6.5.
4. set up the method for umbilical cord stem cell database, it comprises the step of method described in claim 1-3 any one, and following steps: by the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity, and the biological characteristics and the multi-lineage potential that carry out cell are identified, and cell is carried out to molecular genetics diagnosis, preserve all related datas of cell, set up the database of umbilical cord stem cell and carry out associated with freeze-stored cell.
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| CN104983742A (en) * | 2015-04-22 | 2015-10-21 | 南京康雅生物科技有限公司 | Stem cell preparation for treating degenerative osteoarthropathy and preparation method of stem cell preparation |
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| CN111602652A (en) * | 2020-07-03 | 2020-09-01 | 上海中溢精准医疗科技有限公司 | Umbilical cord mesenchymal stem cell cryopreservation protective solution |
| CN112514888A (en) * | 2020-12-02 | 2021-03-19 | 江苏拓弘康恒医药有限公司 | Preparation process of cryopreservation protection solution for umbilical cord mesenchymal stem cells |
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| CN101922048B (en) * | 2010-08-06 | 2014-12-31 | 青岛奥克生物开发有限公司 | Method for constructing public library of umbilical mesenchymal stem cells |
| CN101974484B (en) * | 2010-11-03 | 2013-01-30 | 江苏省北科生物科技有限公司 | Method for preparing human umbilical cord mesenchymal stem cells |
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