CN110317781A - The method of mesenchymal stem cell cryopreserving and recovery - Google Patents

The method of mesenchymal stem cell cryopreserving and recovery Download PDF

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CN110317781A
CN110317781A CN201910651647.3A CN201910651647A CN110317781A CN 110317781 A CN110317781 A CN 110317781A CN 201910651647 A CN201910651647 A CN 201910651647A CN 110317781 A CN110317781 A CN 110317781A
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cell
stem cell
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umbilical cord
storing liquid
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CN110317781B (en
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王小武
迟大明
朱春颖
郭春明
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Tianjin Boya Xiu Yan Biotechnology Co Ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
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    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

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Abstract

The present invention relates to the methods of mesenchymal stem cell cryopreserving and recovery.Mescenchymal stem cell and the method that freezes are prepared the following steps are included: disinfection and cleaning, digestion process, cell culture, cell passage, cells frozen storing liquid is added in mescenchymal stem cell and is frozen in liquid nitrogen.Cells frozen storing liquid includes DMEM-F12, dimethyl sulfoxide and dog blood albumin etc..The method for further relating to freeze mescenchymal stem cell and recover, comprising the following steps: the mescenchymal stem cell being prepared is uniformly mixed with cells frozen storing liquid, then the cell liquid is placed in liquid nitrogen and is freezed, freezes the use until cell；When needing to make cell recovery, the cell frozen is removed from liquid nitrogen, puts into 37 DEG C of water-baths rapidly, melts frozen stock solution completely, cell is transferred on ice bath after melting, and completes cell recovery.Further relate to the cells frozen storing liquid for mesenchymal stem cell cryopreserving and recovery.Excellent technical effect as used in the description is presented in the method for the present invention and frozen stock solution.

Description

The method of mesenchymal stem cell cryopreserving and recovery
Technical field
The invention belongs to stem-cell therapy technical fields, the e.g. stem-cell therapy of canid disease, are related to (from example As dog umbilical cord in) prepare mescenchymal stem cell method and this mescenchymal stem cell treatment such as canid disease Purposes in disease.Excellent technical effect is presented from the method that dog umbilical cord prepares mescenchymal stem cell by the present invention.Between obtained Mesenchymal stem cells can be beneficial treatment canid arthritis, fracture, muscle damage, ligament injury, cartilage damage, joint Damage, cognition dysfunction, immune-mediated disease, xerophthalmia, recurrent uveitis, liver diseases, heart disease, kidney disease Disease, diabetes, enterogastric diseases, thyroid disease, skin disease.Further, the invention further relates to the umbilical cord from dog The method that the mescenchymal stem cell of preparation is frozen, especially, related cell cryopreservation are able to maintain that longer after recovery The Cell viability of time.In addition, mescenchymal stem cell of the present invention can also be from cat；In addition, of the present invention state Mescenchymal stem cell can be fat or umbilical cord from dog or cat.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs or MSC) is present in body interstitial tissue Adult stem cell, can be obtained from a variety of sources such as placenta, umbilical cord, marrow, adipose tissue.MSC has very strong self increase Grow ability and multi-lineage potential.Under controlled condition, it can be divided into vivo or in vitro as nerve cell, cartilage cell, A plurality of types of cells such as fat cell, cardiac muscle cell and osteoblast.MSC also has immune compatibility simultaneously, not will cause Immunological rejection can carry out allograft.In addition MSC does not have oncogenicity also, but can hematopoiesis support, and can secrete more The kind beneficial cells factor and immune factor, therefore regeneration and repairing and treating can be can be safely used for, in traditional medical means beam Huge treatment potential is shown in terms of difficult and complicated cases of the hand without plan.In veterinary applications, stem cell therapy can be effectively improved The quality of life of animal helps them to get rid of ailing puzzlement.Wherein dog is not only important companion animals, can also it is trained at For working dogs such as rescue dogs, seeing-eye dogs, in addition it is also the important animal model of new drug assessment and Preclinical Drug test.Mesh Before, in terms of dog stem-cell therapy, have many by mescenchymal stem cell injection successful treatment dog arthritis, fracture, flesh Meat, ligament or cartilage/joint injury case.Other such as canine cognition dysfunctions, immune-mediated disease, xerophthalmia, Recurrent uveitis, all kinds of liver diseases, heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid gland problem, skin The treatment of disease etc. is also being assessed.
Huge application prospect is there is an urgent need to establish efficient canid mescenchymal stem cell method for separating and preparing, with rule Isolate to modelling high quality MSC.The upper common MSC source of dog is fat and marrow at present, but obtains and do from both sources The process of cell inevitably brings wound and pain to donor animal, and although when placenta/umbilical cord belongs to animal productiong Waste, but they are also one of source abundant MSC, therefore can preferably be protected by them as sample separation cell Protect animal.
CN108456657A (Chinese Patent Application No. 201810278145.6), which is disclosed from the umbilical cord of dog, prepares mesenchyma The method and/or cryopreservation methods of stem cell, method includes the following steps: (1) disinfection and cleaning: with thimerosal to the umbilical cord of dog Tissue surface carries out disinfection, and umbilical cord is cut off, tiling, red on umbilical cord tissue to reduce by buffer solution for cleaning umbilical cord tissue Cell；(2) digestion process: being cut into tissue block for the umbilical cord tissue that step (1) obtains, and tissue block is put into digestion enzyme solutions, Digestion process 0.5-3 hours, after tissue block is removed in filtering, mescenchymal stem cell culture medium is added to terminate digestion, then offsets Change obtained cell and carry out cell cleaning, finally obtains cell suspension；(3) cell culture: the cell suspension that step (2) is obtained It is put into culture vessel, then culture vessel is put into incubator and is cultivated, by culture vessel from culture when culture was to the 2-7 days It is taken out in case, adds appropriate mescenchymal stem cell culture medium, continue to cultivate；At the 8-11 days by culture vessel from incubator It takes out, carries out changing liquid entirely for the first time, continue to cultivate；It carries out within every 1-3 days backward once changing liquid entirely；(4) cell passes on: when culture is held After attached cell fusion rate in device reaches 40%-70%, using digestive ferment by attached cell detachment vessel bottom, it is centrifuged, It takes supernatant away, mesenchymal stem cell media suspension cell again is added, is inoculated in culture vessel and is cultivated, hereafter every 1- It changes the liquid once within 3 days, to get P1 for umbilical cord mesenchymal stem cells after fusion rate reaches 70-90%；Then according to above-mentioned culture side Method carries out necessary passage；Optional (5) freeze: will add cells frozen storing liquid in umbilical cord mesenchymal stem cells obtained by step (4) It is freezed in liquid nitrogen, it is spare.Specifically, a kind of improvement cell jelly comprising following component is disclosed in the CN108456657A Liquid storage: 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of human serum albumins, dextran -40 0.25%w/v are changed using this Good cells frozen storing liquid freezes P1 to P15 for cell, it is stated that frozen-resuscitation process after, the percent survival of cell In 92~96% ranges, being shown in frozen stock solution can be significantly reduced cell cryopreservation-when adding micro dextran and recovered Cell death caused by journey.
Cell recovery is the process opposite with cell cryopreservation, is the process of cell restoration ecosystem, will be frozen in liquid nitrogen Cell in (- 196 DEG C) or refrigerator (dry ice, -60 DEG C~-80 DEG C) is cultivated or is used again after thawing.It is normal when being restored to When temperature state, the morphosis of cell keeps normal, and biochemical reaction can restore.It is different from cell cryopreservation, cell recovery process Heating is fast, prevents the moisture in course of defrosting from entering cell, forms ice crystal, influence cell survival.Cell recovery is usually will Cryovial is removed from liquid nitrogen, and is put into 37 DEG C of water-baths rapidly, is melted frozen stock solution completely in 1~5min, after cell melts 37 DEG C of water-baths are removed as early as possible, and are placed on 4 DEG C of ice baths, and cell recovery is completed；Cell after recovery can then carry out: inspection Survey (such as the detection of the projects such as cell count, survival rate, such as the survival rate of recovery cell can be detected with trypan blue staining), Culture and/or amplification (such as passage), and/or biological applications (such as animal experiment or clinically stem-cell therapy) etc..So And the cell after the recovery of the practical application of stem cell usually requires to place a few hours to tens of hours not equal time, To carry out using preceding other necessary operation sequences, this placement occasionally has at 25 DEG C of temperature, usually in 4 DEG C of temperature conditions Under.Regrettably, it has been found that, being present in the stem cell in existing frozen stock solution, that number is placed at above-mentioned 25 DEG C or 4 DEG C is small The problem of stability deficiency is presented during tens of hours, this kind of Stable Defects are embodied in cell survival rate and survive percentage Number is significantly reduced as standing time extends.
Therefore, this field is urgently expected to have new method to improve the stability of the stem cell after recovery before the use.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing mescenchymal stem cell from the umbilical cord of dog, another mesh of the invention Be, provide a kind of method to improve the stability of the stem cell after recovery before the use, be particularly due in stem cell Practical application recovery after cell usually require to place a few hours to tens of hours not equal time, so as to carry out using Preceding other necessary operation sequences, this placement occasionally have at 25 DEG C of temperature, and usually under the conditions of 4 DEG C of temperature, there are Mr. Yus It is insufficient to that stability can be presented during tens of hours that stem cell in a little frozen stock solutions places a few hours at above-mentioned 25 DEG C or 4 DEG C Problem, this kind of Stable Defects are embodied in cell survival rate i.e. percent survival and are significantly reduced as standing time extends. It has been unexpectedly discovered that preparing mescenchymal stem cell from the umbilical cord of dog using the method for the present invention, encouraging effect is presented Fruit is accomplished the present invention is based on this discovery.In addition, mescenchymal stem cell of the present invention can also be from cat；Separately Outside, mescenchymal stem cell of the present invention of stating can be fat or umbilical cord from dog or cat.
For this purpose, first aspect present invention provides method and/or the side of freezing for preparing mescenchymal stem cell from the umbilical cord of dog Method, method includes the following steps:
(1) it sterilizes and cleans: with thimerosal (such as alcohol, such as 75% ethyl alcohol is as thimerosal) to the umbilical cord tissue of dog (for example, Fresh tissue) surface carries out disinfection, and umbilical cord is cut off, and tiling is (for example, be laid in the culture that diameter is 5-20cm In plate, such as the culture dish of 10cm), pass through buffer (such as PBS buffer solution, such as the 0.025M biphosphate of pH6.5 Sodium buffer) cleaning umbilical cord tissue, to reduce the red blood cell on umbilical cord tissue；(this step be usually in Biohazard Safety Equipment into Row processing)
(2) umbilical cord tissue (in another cell culture plate) that step (1) obtains digestion process: is cut into tissue block (for example, the size of tissue block is about 0.05-0.5 cubic centimetres, preferably about 0.05-0.2 cubic centimetres, preferably from about 0.1 cube Centimetre cube it is blocky), by tissue block be put into digestion enzyme solutions (such as it includes collagenase type Is, DMEM-F12, for example, according to Following method is prepared: the collagenase type I of 0.1g being added to the DMEM-F12 of 100ml, is then obtained by filtration with 0.45 μm of filter Digestive juice) in, digestion process 0.5-3 hours (for example, 37 DEG C digestion process 1-2 hours, such as 1.5 hours), filter removing group After knitting block (for example, being carried out by strainer, the strainer is 50-150 μm of strainer, preferably from about 100 μm of strainers), filled between addition Then matter stem cell media carries out cell cleaning to the cell that digestion obtains, finally obtains cell suspension to terminate digestion； (if not otherwise indicated, the present invention used in mescenchymal stem cell culture medium in the FBS containing 15 parts by weight, 1 parts by weight L- The DMEM-F12 of Glutamine, the Gentamicin of 0.05 parts by weight and 84 parts by weight)
(3) cell culture: by the cell suspension that step (2) obtains be put into culture vessel (for example, with density 0.2-2 × 104/cm2It is added in culture vessel, preferably with density about 1 × 104/cm2It is added), then culture vessel is put into incubator and is carried out Culture, whens culture to the 2-7 days (such as the 3-6 days, such as the 4th day, such as the 5th day), take culture vessel from incubator Out, appropriate (such as 3ml) mescenchymal stem cell culture medium is added, continues to cultivate；It will culture at the 8-11 days (such as the 9th day) Container takes out from incubator, carries out changing liquid entirely for the first time, continues to cultivate；Backward 1-3 days every (such as 2 days) once change entirely Liquid；
(4) cell passes on: after the attached cell fusion rate in culture vessel reaches 40%-70% (such as 60%), Using digestive ferment (for example, in the present invention, if not otherwise specified, using TrypLETMExpress) attached cell is detached from and is held Supernatant is taken in device bottom, centrifugation away, and mesenchymal stem cell media suspension cell again is added, and is inoculated in culture vessel progress Culture, hereafter 1-3 days every (such as every 2 days) change the liquid once, to get P1 after fusion rate reaches 70-90% (such as 80%) For umbilical cord mesenchymal stem cells；Then necessary passage is carried out (for example, TrypLE used in the present invention according to above-mentioned cultural methodTM Express, consisting of: potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, seven water phosphorus Sour disodium hydrogen 2160.0mg/L, EDTA 457.6mg/L, commercialization amount rProtease；The TrypLETMExpress can be with quotient Industry approach is purchased from match Mo Feishier company, and relevant technical information is for example, see http://www.thermofisher.com/ cn/zh/home/technical-resources/media-formulation.346.html)；Optional
(5) it freezes: cells frozen storing liquid will be added in umbilical cord mesenchymal stem cells obtained by step (4) (such as with volume ratio 1:1 Amount be added) freezed in liquid nitrogen, it is spare.(for example, the cells frozen storing liquid includes DMEM-F12, dimethyl sulfoxide and dog blood Albumin.In one embodiment, the cells frozen storing liquid include about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide, About 15 parts of dog blood albumin.
According to method of the first aspect of the present invention, PBS buffer solution described in step (1) is the sodium salt and/or sylvite of phosphoric acid It prepares, pH 5.0-8.0, preferably pH are 5.5-76, and preferably pH is 6.0-7.0.In one embodiment, the PBS The concentration of phosphate radical is 0.01-0.5M, preferably 0.02-0.1M in buffer.In following experiments of the present invention, do not say especially such as Bright, PBS buffer solution used is sodium ascorbyl phosphate, and wherein the concentration of phosphate radical is 0.025M, pH 6.5.It should be noted that this hair Bright people's discovery, PBS buffer solution concentration and pH value within the above range are little for the influential effect of the method for the present invention.
According to method of the first aspect of the present invention, digestion enzyme solutions described in step (2) is that collagenase type I is added DMEM-F12 is obtained by filtration by filter, and digestive ferment 0.05g-0.5g, preferably digestive ferment are 0.08g-0.2g, excellent Selecting digestive ferment is 0.1g, and DMEM-F12 50-500ml, preferably DMEM-F12 are 80-200ml, and preferably DMEM-F12 is 100ml, filter are 0.45 μm of filter.In one embodiment, the digestion enzyme solutions are by the type i collagen of 0.1g Enzyme is added in the DMEM-F12 of 100ml, is mixed, and filtering (such as being filtered with 0.45um filter) obtains.
According to method of the first aspect of the present invention, wherein in step (2), the size of tissue block is about 0.05-0.5 cubes li Rice, preferably about 0.05-0.2 cubic centimetres, preferably from about 0.1 cubic centimetre of cube is blocky.
According to method of the first aspect of the present invention, wherein in step (2), organize block size at 0.05-0.5 cubic centimetres, It is preferred that about 0.1 cubic centimetre of 0.05-0.2 cubic centimetres, especially size when is highly preferred.While it is contemplated that tissue pieces are small Be conducive to the realization of the method for the present invention, however the present inventor is in experiments it is found that in 0.05 cubic centimetre, 0.1 cubic centimetre, 0.5 Under three kinds of states of cubic centimetre, they are almost the same to the digestion process effect of digestive ferment, and volume is greater than after 1 cubic centimetre There is significant adverse effect to the digestion effect of digestive ferment, which can be weak to a certain extent by extending digestion time Change.
According to method of the first aspect of the present invention, wherein in step (2), the time of digestion process is 0.5-3 hours, preferably 1-2.5 hours, preferably 1.5-2 hours.The inventors discovered that disappearing within 1-2.5 hours digestion process time to tissue block Change treatment effect be it is optimal, both can guarantee tissue block obtained sufficient digestion process, be also avoided that cell is destroyed.
According to method of the first aspect of the present invention, wherein in step (2), digestion process is the temperature near body temperature It is carried out in range, preferably 34-40 DEG C, preferably 36-38 DEG C, preferably 37 DEG C.
According to method of the first aspect of the present invention, wherein in step (2), digestion process is carried out in constant-temperature table.
According to method of the first aspect of the present invention, wherein in step (2), tissue block is removed in filtering to be carried out by strainer , the strainer is 50-150 μm of strainer, preferably from about 100 μm of strainers.
According to method of the first aspect of the present invention, wherein in step (2), the mescenchymal stem cell culture medium for terminating digestion is It is added according to the ratio of 2:1~1:2, preferably the ratio of 1:1, the ratio is volume ratio.
According to method of the first aspect of the present invention, wherein in step (2), cell cleaning comprises the concrete steps that 5-15 points of centrifugation Clock removes supernatant, PBS buffer solution is added, cell is resuspended, then be centrifuged 5-15 minutes, remove supernatant, and it is dry thin that mesenchyma is added Born of the same parents' culture medium extracts a small amount of samples and carries out cell count.Centrifugal rotational speed is 800-2000rpm, preferably 1250rpm, centrifugation time It is preferably 10 minutes.
According to method of the first aspect of the present invention, wherein including FBS, L- in the mescenchymal stem cell culture medium Glutamine (Pidolidone), Gentamicin (gentamicin) and DMEM-F12.In one embodiment, it is filled between described Contain the FBS of 10-20% in matter stem cell media.In one embodiment, contain in the mescenchymal stem cell culture medium There is about 15% FBS.In one embodiment, the L- containing 0.5-2% in the mescenchymal stem cell culture medium Glutamine.In one embodiment, about 1% L-Glutamine is contained in the mescenchymal stem cell culture medium.? In one embodiment, the Gentamicin of about 0.01-0.1% is contained in the mescenchymal stem cell culture medium.In a reality It applies in scheme, about 0.05% Gentamicin is contained in the mescenchymal stem cell culture medium.In one embodiment, institute State the DMEM-F12 in mescenchymal stem cell culture medium containing 80-90%.In one embodiment, the mescenchymal stem cell Contain about 84% DMEM-F12 in culture medium.In one embodiment, containing about in the mescenchymal stem cell culture medium The FBS of 15 parts by weight, the L-Glutamine of about 1 parts by weight, the Gentamicin of about 0.05 parts by weight and about 84 parts by weight DMEM-F12。
According to method of the first aspect of the present invention, wherein the mescenchymal stem cell culture medium, which is also augmented, glycine.Example Such as, the FBS containing 15 parts by weight, the L-Glutamine of 1 parts by weight, 0.05 parts by weight in the mescenchymal stem cell culture medium Gentamicin, 84 parts by weight DMEM-F12,0.025%w/v glycine.
According to method of the first aspect of the present invention, wherein the TrypLETMIt is also additionally added in Express digestive ferment 1,2- propylene glycol.For example, the TrypLETMThe 1,2- propylene glycol of 0.05%w/v is also additionally added in Express digestive ferment. It has been had now surprisingly been found that, supplement propylene glycol in glycine and digestive ferment is augmented in mescenchymal stem cell culture medium to be facilitated Improve the yield of mescenchymal stem cell.
According to method of the first aspect of the present invention, wherein also adding dextran -40 in the frozen stock solution, implement at one Further include dextran -40 0.2~0.5%w/v in the frozen stock solution in scheme, preferably also includes 0.25%w/v dextrose Glycosides -40.In one embodiment, which includes about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide, about 15 parts of dog blood albumin, 0.2~0.5%w/v especially 0.25%w/v dextran -40.It has been had now surprisingly been found that, Survival rate when micro dextran -40 helps to improve freeze-stored cell resurrection is added in frozen stock solution.
According to method of the first aspect of the present invention, wherein CO in step (3) described incubator2Concentration is 3-7%, preferably dense Degree is 5%, and incubator temperature controls preferably 34-40 DEG C, preferably 36-38 DEG C, preferably 37 DEG C in body temperature environs. Such CO2Concentration and temperature are usually also condition of culture commonly used in the art.
In addition, it is dry thin to provide separation and amplification mesenchyma from dog umbilical cord flesh tissue in the first aspect of the present invention The method of born of the same parents.Therefore, second aspect of the present invention provides a kind of dog umbilical cord mesenchymal stem cells.
Dog umbilical cord mesenchymal stem cells according to a second aspect of the present invention are any implementation according to a first aspect of the present invention What scheme the method obtained.
Dog umbilical cord mesenchymal stem cells according to a second aspect of the present invention, cell purity are greater than 85%, are greater than 90%.In one embodiment, the umbilical cord mesenchymal stem cells are after 3 more than generation pass on, and cell purity is greater than 85%, example Such as larger than 90%.
Further, third aspect present invention is related to dog umbilical cord mesenchymal stem cells produced by the present invention in preparation for controlling Treat and/or prevention the preparation selected from following canid disease in purposes: arthritis, fracture, muscle damage, ligament injury, Cartilage damage, joint injury, cognition dysfunction, immune-mediated disease, xerophthalmia, recurrent uveitis, liver diseases, Heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid disease, skin disease.
Further, fourth aspect present invention is provided to mescenchymal stem cell (such as any one of first aspect present invention Mescenchymal stem cell made from the method) method that is frozen and recovered, method includes the following steps:
It (51) will be (such as any by digestion process, cell culture and cell succeeding generations, such as first aspect present invention Step (1)~(4) in the method) mescenchymal stem cell that is prepared is uniformly mixed with cells frozen storing liquid, then general The cell liquid, which is placed in liquid nitrogen, to be freezed, and the use until cell is frozen；
(52) when needing to make cell recovery, the cell frozen is removed from liquid nitrogen, is put into 37 DEG C of water-baths rapidly, Melt frozen stock solution completely in 1~5min, cell is transferred on 4 DEG C of ice baths after melting, and completes cell recovery.
Method according to a fourth aspect of the present invention, wherein mescenchymal stem cell and cells frozen storing liquid are with volume ratio 1:1 Ratio mixing.
Method according to a fourth aspect of the present invention, wherein the cells frozen storing liquid includes 65 parts of DMEM-F12,10 parts of diformazans Base sulfoxide, 15 parts of dog blood albumin, 0.2~0.5%w/v especially 0.25%w/v dextran -40.
Method according to a fourth aspect of the present invention, wherein the cells frozen storing liquid includes 65 parts of DMEM-F12,10 parts of diformazans Base sulfoxide, 15 parts of dog blood albumin, 0.03~0.05%w/v such as 0.04%w/v PLURONICS F87 and 0.5~0.7%w/v Such as 0.6%w/v histidine.Unexpected hair flesh not only can when both poloxamer and histidine are applied in combination It is enough effectively maintain cell freeze-resuscitation process in percent survival, and after cell recovery in the case where not changing liquid It is able to maintain that the high viability of long period maintains excellent survival stability.
Method according to a fourth aspect of the present invention, wherein the cells frozen storing liquid is prepared according to such as under type: will provide DMEM-F12, dimethyl sulfoxide, the dog blood albumin of ratio are uniformly mixed by volume, and the PLURONICS F87 of specified amount is added And histidine, make dissolution to get.The preparation method is the preparation method of this field routine, is related to frozen stock solution in the present invention When, it is that using similar approach is prepared if not otherwise specified.
Method according to a fourth aspect of the present invention, wherein the mesenchyma that the mescenchymal stem cell is dog umbilical cord source is done carefully Born of the same parents.
Further, fifth aspect present invention provides that (such as first aspect present invention is any for mescenchymal stem cell Mescenchymal stem cell made from the method) cells frozen storing liquid that freezes and recover, wherein including 65 parts of DMEM-F12,10 parts Dimethyl sulfoxide, 15 parts of dog blood albumin, 0.03~0.05%w/v such as 0.04%w/v PLURONICS F87s and 0.5~0.7% W/v such as 0.6%w/v histidine.
Cells frozen storing liquid according to a fifth aspect of the present invention is to prepare according to such as under type: by the DMEM- of regulated proportion F12, dimethyl sulfoxide, dog blood albumin are uniformly mixed by volume, and the PLURONICS F87 and histidine of specified amount is added, makes Dissolution to get.
Cells frozen storing liquid according to a fifth aspect of the present invention, wherein the mescenchymal stem cell freeze and recovery include with Lower step:
It (51) will be (such as any by digestion process, cell culture and cell succeeding generations, such as first aspect present invention Step (1)~(4) in the method) mescenchymal stem cell that is prepared is uniformly mixed with cells frozen storing liquid, then general The cell liquid, which is placed in liquid nitrogen, to be freezed, and the use until cell is frozen；
(52) when needing to make cell recovery, the cell frozen is removed from liquid nitrogen, is put into 37 DEG C of water-baths rapidly, Melt frozen stock solution completely in 1~5min, cell is transferred on 4 DEG C of ice baths after melting, and completes cell recovery.
Cells frozen storing liquid according to a fifth aspect of the present invention, wherein mescenchymal stem cell and cells frozen storing liquid are with volume ratio The ratio mixing of 1:1.
Either side according to the present invention, wherein the mescenchymal stem cell can also be from cat.
Either side according to the present invention, wherein the mescenchymal stem cell can be fat or navel from dog or cat Band.
Either side according to the present invention, wherein the source for mesenchymal stem cells is when the fat or umbilical cord of cat, it is used Dog blood albumin replace with cat blood albumin.
The present invention is further illustrated below.Cited text in document cited in the present invention and the document It offers, their full content is incorporated herein by reference.
In the present invention, in any technical solution of either side of the present invention, any technical characteristic is equally applicable to this Any embodiment in either invention face, as long as they will not cause contradiction, and this be mutually applicable in if necessary may be used To be suitably modified.
In the present invention, term " umbilical cord mesenchymal stem cells " refers to the mescenchymal stem cell from umbilical cord.Therefore exist In the present invention, more particularly in context of the invention, term " umbilical cord mesenchymal stem cells " can with " umbilical cord stem cells ", " stem cell ", " mescenchymal stem cell " are used interchangeably, unless otherwise clearly indicating.
In the present invention, term " PBS buffer solution " or " PBS " refer to phosphate buffer.Those skilled in the art are ripe Know the general formula and preparation method and their general aspects such as pH value or pH of the PBS used under situation of the present invention Range.
In the present invention, term " umbilical cord " refers to the umbilical cord of dog, particularly relates to the umbilical cord within 4 hours postpartum.
It is filled between mescenchymal stem cell (mesenchymal stem cell, MSC) such as the mammal such as mankind or dog Matter stem cell is separated from marrow earliest, from mesoblastic a kind of with multi-lineage potential and self-renewing The tissue stem cell of ability has under external specified conditions to osteoblast, cartilage cell, fat cell, endothelium in vivo Ability (the Caplan AI.Mesenchymal of a variety of adult cell differentiation such as cell, nerve cell, myocyte, liver cell 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).It is newest research shows that mescenchymal stem cell has immunological regulation and Hematopoiesis Support affect, and be easy to outer The expression of source channel genes.Therefore mescenchymal stem cell not still tissue-engineered bone, cartilage and cardiac muscle building in seed cell, Important carrier cell in gene therapy, and since mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host of inhibition of transplant anti- Function is answered, is with a wide range of applications in hematopoietic stem cell transplantation and organ transplant.Mescenchymal stem cell has external patch The characteristic of wall growth, using this characteristic, people have succeeded from liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood Mescenchymal stem cell is separately cultured out in equal Various Tissues.As commonly understood in the art, in the present invention, such as not in addition Illustrate, dog blood albumin used is the dog blood albumin of 10% concentration.In the present invention, DMEM-F12 used can be easy It is obtained from a variety of brands of commercially available approach, in the present invention if not otherwise specified, uses the DMEM-F12 of Gibco brand. In the present invention, it when referring to " freezing ", if not otherwise specified, each means and is frozen in liquid nitrogen, this to freeze operation be this The routine operation in field, specific operation process is for example are as follows: mixes the mescenchymal stem cell being prepared with cells frozen storing liquid It is even, then the cell liquid is placed in liquid nitrogen and is freezed, the use until cell is frozen；
In the present invention, when referring to " recovery ", if not otherwise specified, the conventional resuscitation of this field is each meant, specifically Operating process is for example are as follows: when needing to make cell recovery, the cell frozen is removed from liquid nitrogen, puts into 37 DEG C of water-baths rapidly In, melt frozen stock solution completely in 1~5min, cell is transferred on 4 DEG C of ice baths after melting, and completes cell recovery.
The method of the invention discloses a kind of from umbilical cord a large amount of separating mesenchymal stem cells, and protected using this method Storing umbilical mesenchymal stem cells simultaneously establish umbilical cord stem cells library.The present inventor was separately cultured mesenchyma in summary in the past and did On the basis of cell, using tissue digestion enzymic digestion umbilical cord tissue block, in conjunction with stationary culture, success is isolated from umbilical cord A large amount of mescenchymal stem cells.The mescenchymal stem cell that the method for the present invention obtains is with high purity, quantity is more, has dry with medulla mesenchyma The identical biological characteristics of cell can break up to osteoblast, cartilage cell, fat cell, endothelial cell, nerve cell etc.. Since stem cell is inmature compared with adult stem cell in umbilical cord, rich content is clinically with a wide range of applications, we use Conventional cell freezing method freezes mescenchymal stem cell as bleeding of the umbilicus, establishes umbilical cord stem cells library, is later dry The further investigation of cell and clinical treatment lay the foundation.
Due to candidate stem cell rich in bleeding of the umbilicus, people are this important biology of the umbilical cord candidate stem cell of dog Resource stores, and provides a kind for the treatment of means for a variety of diseases in the blood system and disease of immune system.Same umbilical cord mesenchyma As a kind of more importantly stem cell resource, we are frozen in -196 with conventional cell freezing method and are taken the photograph stem cell It is saved for a long time in the profound hypothermia liquid nitrogen of family name's degree, establishes umbilical cord stem cells library, be stem cell in the future or application for the treatment of preservation seed.
According to the method for the present invention, in-between mesenchymal stem cell media, which is matched, to succeed and effectively to umbilical cord mesenchyma Stem cell carries out amplification in vitro.According to the method for the present invention, wherein changing liquid and to organize the setting of checkout time to shorten adherent thin Born of the same parents reach the time of specified fusion rate.According to the method for the present invention, the formula of digestive ferment and the digestion time of umbilical cord tissue and side Method can succeed and effectively the full cell in tissue is separated.
Operation of the present invention is simple, convenient and practical, can obtain a large amount of mescenchymal stem cell, and differentiation performance is good, have at The ability of the cell differentiations such as osteocyte, fat cell, cartilage cell, endothelial cell, nerve cell.Navel of the present invention success from dog Separation obtains a large amount of higher mescenchymal stem cells of purity in band, and can establish the umbilical cord stem cells library of dog with this method to store up The stem cell of standby this great application prospect.The method is simple and easy to do, and since umbilical cord is as bleeding of the umbilicus, Cell Component is inmatureer, It is from a wealth of sources, it is conveniently easy to get, therefore method of the invention will above have extensive prospect in the canid application of stem cell.
Specific embodiment
The present invention can be further described by the following examples, however, the scope of the present invention and unlimited In following embodiments.One of skill in the art, can be with it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Various change and modification are carried out to the present invention.The present invention carries out the material and test method arrived used in test general And/or specific description.Although to realize the present invention many materials and operating method used in purpose be it is known in the art that But the present invention still makees description as detailed as possible herein.
Embodiment 1 prepares mescenchymal stem cell from the umbilical cord of dog
(1) sterilize and clean: in Biohazard Safety Equipment, with thimerosal (75% ethyl alcohol) to the umbilical cord tissue of dog (it is fresh from Somatic umbilicus tissue) surface carries out disinfection, umbilical cord cut off, and tiling (is laid in the culture dish that diameter is 10cm), passes through buffering Liquid (the 0.025M phosphate sodium dihydrogen buffer solution of pH6.5) cleans umbilical cord tissue, to reduce the red blood cell on umbilical cord tissue；
(2) umbilical cord tissue that step (1) obtains digestion process: is cut into tissue block in another cell culture plate Tissue block is put into digestion enzyme solutions (preparation method: by the collagenase type I of 0.1g by (about 0.1 cubic centimetre of cube is blocky) The DMEM-F12 of 100ml is added, digestive juice then is obtained by filtration with 0.45 μm of filter) in, 0.5-3 hours (these of digestion process In example, 37 DEG C of digestion process 1.5 hours), after tissue block (this example passes through 100 μm of strainers and carries out) is removed in filtering, mesenchyma is added Stem cell media (is added with the ratio of volume ratio 1:1；In this example, 15 parts by weight are contained in mescenchymal stem cell culture medium The DMEM-F12 of FBS, the L-Glutamine of 1 parts by weight, the Gentamicin of 0.05 parts by weight and 84 parts by weight) disappeared with terminating Change, then carrying out cell cleaning to the cell that digestion obtains, (in this example, 1250rpm is centrifuged 10 minutes removal supernatants, is added PBS buffer solution is resuspended after cell again 1250rpm and is centrifuged 10 minutes removal supernatants, and addition mescenchymal stem cell culture medium (extracts A small amount of samples carry out cell count)), finally obtain cell suspension；
(3) cell culture: by the cell suspension that step (2) obtains be put into culture vessel (in this example, with density about 1 × 104/cm2It is added), then put culture vessel into incubator (CO2Concentration is 5%, 37 DEG C of temperature) in cultivated, culture to the Culture vessel is taken out from incubator when 2-7 days (this example, general culture was to the 4th day), it is dry thin to add appropriate (3ml) mesenchyma Born of the same parents' culture medium continues to cultivate；Culture vessel is taken out from incubator at the 8-11 days (this example, the 9th day), is carried out for the first time Liquid is changed entirely, continues to cultivate；Backward 1-3 days every (this example, 2 days) carries out once changing liquid entirely；
(4) cell passes on: after the attached cell fusion rate in culture vessel reaches 40%-70% (this example, 60%), Using digestive ferment, (this example uses TrypLETMExpress) by attached cell detachment vessel bottom, centrifugation is taken supernatant away, is added Enter mesenchymal stem cell media suspension cell again, is inoculated in culture vessel and is cultivated, hereafter 1-3 days every (this example, every 2 It) it changes the liquid once, until fusion rate reaches after 70-90% (this example up to 80%) to get P1 for umbilical cord mesenchymal stem cells；Then Necessary passage, which is carried out, according to above-mentioned cultural method (in this example, P15 generation is passaged to, wherein cell purity is greater than after the passage of 3 generations 95%；This example, commercial prod TrypLE usedTMExpress is consisting of: potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, seven water disodium hydrogen phosphate 2160.0mg/L, EDTA457.6mg/L, commercialization amount rProtease；The TrypLETMExpress can be by commercial sources purchased from match Mo Feishier company, relevant technical information Referring to http://www.thermofisher.com/cn/zh/home/technical-resources/medi a- formulation.346.html)；
(5) it freezes: cells frozen storing liquid (volume ratio 1:1) will be added in umbilical cord mesenchymal stem cells obtained by step (4), in liquid It is freezed in nitrogen, spare (this example, cells frozen storing liquid used include 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 portions of white eggs of dog blood It is white).Before freezing, by the details of mescenchymal stem cell (including dog age, kind of dog, hereditary information, immunization campaign information, disease Malicious detection information etc.) input computer data, it is for future reference to establish data archival.
Embodiment 2 prepares mescenchymal stem cell from the umbilical cord of dog
(1) sterilize and clean: in Biohazard Safety Equipment, with thimerosal (75% ethyl alcohol) to the umbilical cord tissue of dog (it is fresh from Somatic umbilicus tissue) surface carries out disinfection, umbilical cord cut off, and tiling (is laid in the culture dish that diameter is 10cm), passes through buffering Liquid (the 0.025M phosphate sodium dihydrogen buffer solution of pH6.5) cleans umbilical cord tissue, to reduce the red blood cell on umbilical cord tissue；
(2) umbilical cord tissue that step (1) obtains digestion process: is cut into tissue block in another cell culture plate Tissue block is put into digestion enzyme solutions (preparation method: by the collagenase type I of 0.1g by (about 0.1 cubic centimetre of cube is blocky) The DMEM-F12 of 100ml is added, digestive juice then is obtained by filtration with 0.45 μm of filter) in, 0.5-3 hours (these of digestion process In example, 37 DEG C of digestion process 1.5 hours), after tissue block (this example passes through 100 μm of strainers and carries out) is removed in filtering, mesenchyma is added Stem cell media (is added with the ratio of volume ratio 1:1；In this example, 15 parts by weight are contained in mescenchymal stem cell culture medium FBS, the L-Glutamine of 1 parts by weight, the Gentamicin of 0.05 parts by weight, 84 parts by weight DMEM-F12,0.025%w/v Glycine) to terminate digestion, then carrying out cell cleaning to the cell that digestion obtains, (in this example, 1250rpm is centrifuged 10 minutes and goes Except supernatant, PBS buffer solution is added, 10 minutes removal supernatants of 1250rpm centrifugation again are resuspended after cell, it is dry thin that mesenchyma is added Born of the same parents' culture medium (extract a small amount of samples and carry out cell count)), finally obtain cell suspension；
(3) cell culture: by the cell suspension that step (2) obtains be put into culture vessel (in this example, with density about 1 × 104/cm2It is added), then put culture vessel into incubator (CO2Concentration is 5%, 37 DEG C of temperature) in cultivated, culture to the Culture vessel is taken out from incubator when 2-7 days (this example, general culture was to the 4th day), it is dry thin to add appropriate (3ml) mesenchyma Born of the same parents' culture medium continues to cultivate；Culture vessel is taken out from incubator at the 8-11 days (this example, the 9th day), is carried out for the first time Liquid is changed entirely, continues to cultivate；Backward 1-3 days every (this example, 2 days) carries out once changing liquid entirely；
(4) cell passes on: after the attached cell fusion rate in culture vessel reaches 40%-70% (this example, 60%), Using digestive ferment, (this example uses TrypLETMExpress is also additionally added to 1, the 2- the third two of 0.05%w/v in the digestive ferment Alcohol) by attached cell detachment vessel bottom, supernatant is taken in centrifugation away, mesenchymal stem cell media suspension cell again is added, then It is inoculated in culture vessel to be cultivated, hereafter 1-3 days every (this example, every 2 days) changes the liquid once, until fusion rate reaches 70-90% To get P1 for umbilical cord mesenchymal stem cells after (this example up to 80%)；Then necessary passage (this example is carried out according to above-mentioned cultural method In, it is passaged to P15 generation, wherein cell purity is greater than 95% after the passage of 3 generations；This example, TrypLE usedTMIts composition of Express Are as follows: potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, seven water disodium hydrogen phosphates 2160.0mg/L, EDTA 457.6mg/L, commercialization amount rProtease；The TrypLETMExpress can pass through business way Diameter is purchased from match Mo Feishier company, and relevant technical information is referring to http://www.thermofisher.com/cn/zh/ home/technical-resources/media-formulation.346.html)；
(5) it freezes: cells frozen storing liquid (volume ratio 1:1) will be added in umbilical cord mesenchymal stem cells obtained by step (4), in liquid It is freezed in nitrogen, spare (this example, cells frozen storing liquid used include 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 portions of white eggs of dog blood It is white).Before freezing, by the details of mescenchymal stem cell (including dog age, kind of dog, hereditary information, immunization campaign information, disease Malicious detection information etc.) input computer data, it is for future reference to establish data archival.
In embodiment 1, from step (1) to step (4), gained P1 is tested for dog umbilical cord mesenchymal stem cells through 5 times, The average yield of every gram of umbilical cord tissue is 3~7 × 105(in the same method of the embodiment of the present invention 1 to people's umbilical cord in cell context When tissue is handled, the average yield of every gram of umbilical cord tissue can reach 3~6 × 107Cell context, big two orders of magnitude, this It may be as caused by species variation).In the present embodiment 2, from step (1) to step (4), gained P1 is filled between dog umbilical cord Matter stem cell, the average yield of every gram of umbilical cord tissue is 4~9 × 107In cell context (average result tested through 5 times, under Together).In the complementary testing referring to embodiment 2, glycine is not augmented in mescenchymal stem cell culture medium, then P1 is for dog umbilical cord The average yield of every gram of umbilical cord tissue of mescenchymal stem cell is 2~4 × 105Cell context.It is tried in the supplement referring to embodiment 2 In testing, in digestive ferment TrypLETM1,2-PD is not augmented in Express, then every gram for dog umbilical cord mesenchymal stem cells of P1 The average yield of umbilical cord tissue is 3~5 × 105Cell context；This shows only to augment sweet ammonia in mescenchymal stem cell culture medium Acid, simultaneously in digestive ferment TrypLETMWhen augmenting 1,2-PD in Express, it is dry can just to significantly improve dog umbilical cord mesenchyma The yield of cell.
In the present invention, if not otherwise indicated, the platform of this field routine is all made of when measuring cell number and/or cell viability Expect blue decoration method.
Make 1~2 gained dog umbilical cord mesenchymal stem cells conventional cryopreservation liquid of above embodiments of the present invention (formula are as follows: 65 parts DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin) it freezes 30 days, it is then recovered with conventional method, measurement is frozen The cell number after cell number and recovery before depositing, with cell number after recovery divided by born of the same parents' number before freezing multiplied by 100% gained hundred Score is as percent survival.1 gained P1 of embodiment to being frozen of P15 generation-resuscitation process percent survival 67~ 75% range, for example, embodiment 1 P3 for dog umbilical cord mesenchymal stem cells percent survival be 72%；2 gained of embodiment P1 to being frozen of P15 generation-resuscitation process percent survival in 65~76% ranges, such as embodiment 2 P5 for dog umbilical cord The percent survival of mescenchymal stem cell is 68%.
Freezing and recovering for the mescenchymal stem cell of this field routine is implemented according to the method for including step: will be prepared into The mescenchymal stem cell arrived is uniformly mixed with cells frozen storing liquid, and then the cell liquid is placed in liquid nitrogen and is freezed, and is frozen until thin The use of born of the same parents；When needing to make cell recovery, the cell frozen is removed from liquid nitrogen, is put into 37 DEG C of water-baths rapidly, 1~ Melt frozen stock solution completely in 5min, cell is transferred on 4 DEG C of ice baths after melting, and completes cell recovery.In invention, such as not In addition illustrate, freezing and recovering for being related to is implemented according to the above method.
Embodiment 3: referring to embodiment 1, but in step (5), using improvement cells frozen storing liquid instead (wherein includes: 65 parts DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.25%w/v dextrans -40), P1 to P15 as the result is shown Frozen for cell-percent survival of resuscitation process in 92~96% ranges, such as P3 is for dog umbilical cord mesenchymal stem cells Percent survival is 93%.
Embodiment 4: referring to embodiment 2, but in step (5), using improvement cells frozen storing liquid instead (wherein includes: 65 parts DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.25%w/v dextrans -40), P1 to P15 as the result is shown Frozen for cell-percent survival of resuscitation process in 91~97% ranges, such as P5 is for dog umbilical cord mesenchymal stem cells Percent survival is 94%.It has now surprisingly been found that, can significantly be improved when adding micro dextran in frozen stock solution Cell death caused by cell cryopreservation-resuscitation process.
In the present invention, the various cell cryopreservation liquid making methods being related to if not otherwise indicated are prepared according to following method : the DMEM-F12 of regulated proportion, dimethyl sulfoxide, dog blood albumin are uniformly mixed by volume, are optionally added specified amount Other specified amounts component make dissolution to get.
Embodiment 5: referring to embodiment 1, but in step (5), using another improvement cells frozen storing liquid instead, (it be can be described as Cells frozen storing liquid B1, wherein including: 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.04%w/v pool Luo Shamu 188 and 0.6%w/v histidine), it recovers after 30 days are frozen in liquid nitrogen, P1 to P15 is passed through for cell as the result is shown Freeze-percent survival of resuscitation process in 93~96% ranges, such as P3 is for the survival percentage of dog umbilical cord mesenchymal stem cells Number is 94%.
Embodiment 6: referring to embodiment 2, but in step (5), using another improvement cells frozen storing liquid instead, (it be can be described as Cells frozen storing liquid B1, wherein including: 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.04%w/v pool Luo Shamu 188 and 0.6%w/v histidine), it recovers after 30 days are frozen in liquid nitrogen, P1 to P15 is passed through for cell as the result is shown Freeze-percent survival of resuscitation process in 93~97% ranges, such as P5 is for the survival percentage of dog umbilical cord mesenchymal stem cells Number is 95%.
Embodiment 7: referring to embodiment 1, but in step (5), using another improvement cells frozen storing liquid instead, (it be can be described as Cells frozen storing liquid B2, wherein including: 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.03%w/v pool Luo Shamu 188 and 0.7%w/v histidine), it recovers after 30 days are frozen in liquid nitrogen, P1 to P15 is passed through for cell as the result is shown Freeze-percent survival of resuscitation process in 92~96% ranges, such as P3 is for the survival percentage of dog umbilical cord mesenchymal stem cells Number is 93%.
Embodiment 8: referring to embodiment 1, but in step (5), using another improvement cells frozen storing liquid instead, (it be can be described as Cells frozen storing liquid B3, wherein including: 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.05%w/v pool Luo Shamu 188 and 0.5%w/v histidine), it recovers after 30 days are frozen in liquid nitrogen, P1 to P15 is passed through for cell as the result is shown Freeze-percent survival of resuscitation process in 94~96% ranges, such as P3 is for the survival percentage of dog umbilical cord mesenchymal stem cells Number is 95%.
Stability after experimental example 1, freeze-stored cell recovery
The cell recovered after freezing Examples 1 to 8 in liquid nitrogen 30 days using corresponding frozen stock solution, the case where not changing liquid Under, placed 8 hours at 25 ± 1 DEG C of temperature, measured respectively using trypan blue staining cell this 25 DEG C -8 hours disposition before, The viable count in unit volume cell liquid afterwards, with viable count at 8 hours divided by quotient obtained by viable count at 0 hour again It is remaining percentage of the cell after 25 DEG C of disposition in -8 hours multiplied by 100% gained percentage.As a result, Examples 1 to 4 cell Remaining percentage is respectively 42.3%, 45.6%, 38.7%, 44.1%；5~8 cell rests percentage of embodiment is respectively 89.5%, 91.2%, 88.4%, 86.7%.
Stability after experimental example 2, freeze-stored cell recovery
The cell recovered after freezing Examples 1 to 8 in liquid nitrogen 30 days using corresponding frozen stock solution, the case where not changing liquid Under, placed 96 hours at 4 ± 1 DEG C of temperature, measured respectively using trypan blue staining cell this 4 DEG C -96 hours disposition before, The viable count in unit volume cell liquid afterwards, with viable count at 96 hours divided by quotient obtained by viable count at 0 hour It is remaining percentage of the cell after 4 DEG C of disposition in -96 hours multiplied by 100% gained percentage.As a result, Examples 1 to 4 is thin Born of the same parents remnants' percentage is respectively 47.4%, 44.5%, 52.3%, 42.7%；5~8 cell rests percentage of embodiment is respectively 91.7%, 89.4%, 93.2%, 88.6%.
According to the result of above-mentioned experimental example 1-2 as it can be seen that the cell after recovery is not when using cells frozen storing liquid B1~B3 It changes under the conditions of liquid, can be significantly than the cell survival longer time in other frozen stock solutions, this will provide suitable for mescenchymal stem cell Answering property significantly stronger use occasion and use environment.
The embodiment 9 of supplement: respectively referring to embodiment 5~8, but be not added with poloxamer in cells frozen storing liquid used, Cell is recovered after freezing 30 days, refers again to method measurement/calculating remnants percentage of experimental example 1；As a result, respectively referring to Embodiment 5~8 but cell rests percentage is respectively 44.3%, 41.8%, 46.2%, 39.7% when being not added with poloxamer. The embodiment 10 of supplement: embodiment 5~8 is respectively referred to, but is not added with histidine in cells frozen storing liquid used, cell is freezing It recovers after 30 days, refers again to method measurement/calculating remnants percentage of experimental example 1；As a result, respectively referring to embodiment 5~8 But cell rests percentage is respectively 40.6%, 37.3%, 44.6%, 39.3% when being not added with histidine.The embodiment of supplement 11: respectively referring to embodiment 5~8, but be not added with poloxamer in cells frozen storing liquid used, cell carries out after freezing 30 days Recovery, refers again to method measurement/calculating remnants percentage of experimental example 2；As a result, respectively referring to embodiment 5~8 but being not added with pool Cell rests percentage is respectively 38.7%, 44.3%, 42.7%, 41.5% when Luo Shamu.The embodiment 12 of supplement: join respectively According to embodiment 5~8, but histidine is not added in cells frozen storing liquid used, cell is recovered after freezing 30 days, is referred again to The method measurement of experimental example 2/calculating remnants percentage；As a result, cell when respectively referring to embodiment 5~8 but being not added with histidine Remaining percentage is respectively 47.3%, 42.6%, 44.1%, 42.2%.According to these results as it can be seen that cell is after recovery, only Excellent stability is just presented when simultaneously including poloxamer and histidine in cells frozen storing liquid.
The Identification of Biological Characteristics of experimental example 3, umbilical cord MSC
Following tests, the test of two kinds of stem cells are carried out to embodiment 1 and 2 gained dog umbilical cord mesenchymal stem cells of embodiment As a result indifference.
1, cell growth and its Morphological Characteristics
By being separately cultured for embodiment 1 and embodiment 2, aobvious after gained dog umbilical cord mesenchymal stem cells culture 48 hours Shuttle shape attached cell can be obviously seen under micro mirror, be will form within 8 days or so turbine-like cell clone, be will form 70% after had digestive transfer culture The leapfrog structure of left and right fusion.In incubation, it is found that this cellular morphology is relatively uniform, growth rate is fast, and adherent speed is fast, easily It is digested by pancreatin, is passaged to 3-15 generation, form and growth characteristic are also without substantially changeing.
2, flow cytometry identification of M SC surface marker
The 1st, 3,5,10,15 generation cells, Flow cytometry cell surface marker, dynamic observation incubation are taken respectively The variation of middle cell surface marker.Cell phenotype testing result show the distinctive mark CD44 of cell statement mescenchymal stem cell, CD90, CD105 do not express CD11, CD19, CD34.
3, the drafting of umbilical cord MSC growth curve and the measurement of logarithmic growth phase doubling time
Logarithmic growth phase cell, digestion count, with the LG-DMEM culture medium of 10%FBS be made cell suspension (2 × 104/ ml), every hole is inoculated with 0.5ml in 24 orifice plates, and 37 DEG C, 5%CO2, cultivate under saturated humidity.3 multiple holes, trypan blue dye are taken daily Living cell counting number after color calculates average value, is observed continuously 7 days.Using incubation time as horizontal axis, cell number is the longitudinal axis, is drawn thin Intracellular growth curve.Cell is calculated in the doubling time of logarithmic growth phase, i.e. Td=Tlg2/Lg (Nt/ with Patterson formula No), Td: the doubling time (h), T: cell increases to the time (h) used in Nt as No, N: cell number.
Cell growth curve is drawn by the result of daily cell count, calculates the doubling time.It can by cell growth curve To find out, cell was in exponential phase of growth at the 3-5 days.The 5th generation cell is calculated from the formula in the multiplication of exponential phase of growth Time is in 22-36 hours ranges.
4, the identification of umbilical cord MSC multi-lineage potential
(1) osteogenic induction: the 3 generations above MSC, by 5 × 104/ hole is inoculated with six orifice plates, is put in 37 DEG C, 5%CO2, saturated humidity Under, after being cultivated for 24 hours in MSC culture medium, uses instead and screened the DMEM-HG of FBS containing 10% and 0.1 μM of dexamethasone, anti-bad is added 50 μM of hematic acid phosphate, β-phosphoglycerol 5mM, are put in 37 DEG C, 5%CO2, cultivate under saturated humidity, every 3 days half amounts change liquid, Coinduction 2-4 weeks.Alkaline phosphatase staining identification osteoblast is formed, and Von Kossa dyeing identification bone tubercle is formed.Containing 10% is screened the DMEM-HG of FBS, and 0.1 μM of dexamethasone, 50 μM of ascorbyl phosphate, β-phosphoglycerol 5mM culture is added 1 week, apparent change occurred for cellular morphology, becomes polygonal from fusiform fibroblast sample, was similar to neuronal cell Sample, it is prominent that long filiform occurs in cell periphery, and can extend to surrounding.After continuing culture 2 weeks or more, occurs calcification in cellular matrix Spot, mineralizer gradually appear, and initially form the small junction structure of multilayer, until after culture 4 weeks, it is seen that obvious calcium scoring.At 2 weeks Alkaline phosphatase staining is in strong positive reaction, reaches 92% or more, and the control group not induced is largely then feminine gender, only Have and be shown as weakly positive less than 5%, shows that cell is converted to osteoblast.Von Kossa dyeing can will deposit in bone tubercle Calcium dye black, the visible a large amount of black bone tubercle of induction group has an apparent stereochemical structure, and control group is at any time all There is no positive reaction.
(2) Adipogenic induction: the 3 generations above MSC, by 5 × 104/ hole is inoculated in six orifice plates, is put in 37 DEG C, 5%CO2, saturation Under humidity, after being cultivated for 24 hours in MSC culture medium, uses instead and screened the DMEM in high glucose of FBS containing 10%, and dexamethasone 0.5 is added μM, 25 μM of indocin, IBMX 0.5mM, 2 μ g/ml of insulin, be put in 37 DEG C, 5%CO2, cultivate under saturated humidity, every 3 days half Amount changes liquid, and coinduction 2 weeks, oil red dyeing identification fat drips were formed., through screening the DMEM-HG of FBS, dexamethasone is being added containing 10% 0.5 μM, 50 μM of indocin, IBMX 0.5mM, 5 μ g/ml of insulin culture 3 days, morphologic change occurs for cell, by fusiform Fibroblast sample, which gradually tapers up, to shorten, and 90% or more cell becomes cube or polygonal；Continuous culture 7 days, it is visible under mirror There are small fat drips to occur into the cell, with the extension of incubation time, fat drips are gradually increased and merge, until when cultivating 2 weeks, it is seen that melt Pockets of fat drips are closed full of entire cell.The fat generated in oil red O stain visible cell dyes red by specificity.
The dog umbilical cord mesenchymal stem cells recovered after freezing 30 days to embodiment 3~8 carry out above-mentioned test, as a result with reality Apply the test result indifference of two kinds of stem cells of example 1 and embodiment 2.
By the detection of above series of data target, show dry using the isolated dog mesenchyma of the method for the present invention Cell has the ability to differentiation such as osteoblast, fat cells, it was demonstrated that the dog mescenchymal stem cell tool that the method for the present invention obtains There are stem cell properties.
The present inventor is in the test of supplement, referring to each embodiment and experiment of hereinbefore " specific embodiment " part Example, the difference is that use dog umbilical cord therein instead dog fat, it is identical like that with above-mentioned dog umbilical cord result, it equally can be effectively Dog fat mesenchymal stem cell is obtained, and such as above-mentioned each experiment is similarly presented in these obtained dog fat mesenchymal stem cells Performance as example.
The present inventor is in the test of supplement, referring to each embodiment and experiment of hereinbefore " specific embodiment " part Example, the difference is that use dog umbilical cord therein instead cat umbilical cord, it is identical like that with above-mentioned dog umbilical cord result, it equally can be effectively Cat umbilical cord mesenchymal stem cells are obtained, and such as above-mentioned each experiment is similarly presented in these obtained cat umbilical cord mesenchymal stem cells Performance as example.
The present inventor is in the test of supplement, referring to each embodiment and experiment of hereinbefore " specific embodiment " part Example, the difference is that use dog umbilical cord therein instead cat fat, it is identical like that with above-mentioned dog umbilical cord result, it equally can be effectively Cat fat mesenchymal stem cell is obtained, and such as above-mentioned each experiment is similarly presented in these obtained cat fat mesenchymal stem cells Performance as example.
This method is easy to operate, stablizes, and fat or the umbilical cord mesenchymal stem cells activity being thus prepared are high, can be used for The treatment of dog or cat conventional means refractory disease.

Claims (10)

1. from the method that the umbilical cord of dog prepares mescenchymal stem cell and freezes, method includes the following steps:
(1) it sterilizes and cleans: being carried out disinfection with umbilical cord tissue surface of the thimerosal to dog, umbilical cord is cut off, tile, pass through buffering Liquid cleans umbilical cord tissue, to reduce the red blood cell on umbilical cord tissue；
(2) digestion process: being cut into tissue block for the umbilical cord tissue that step (1) obtains, and tissue block is put into digestion enzyme solutions, is disappeared Change processing 0.5-3 hours, after tissue block is removed in filtering, mescenchymal stem cell culture medium is added to terminate digestion, then to digestion Obtained cell carries out cell cleaning, finally obtains cell suspension；
(3) cell culture: the cell suspension that step (2) obtains is put into culture vessel, then culture vessel is put into incubator It is cultivated, culture vessel is taken out from incubator when culture was to the 2-7 days, adds appropriate mescenchymal stem cell culture medium, Continue to cultivate；Culture vessel is taken out from incubator at the 8-11 days, carries out changing liquid entirely for the first time, continues to cultivate；Backward It carries out within every 1-3 days once changing liquid entirely；
(4) cell passes on: after the attached cell fusion rate in culture vessel reaches 40%-70%, will be pasted using digestive ferment Supernatant is taken in parietal cell detachment vessel bottom, centrifugation away, and mesenchymal stem cell media suspension cell again is added, is inoculated in Culture vessel is cultivated, and is changed the liquid once within hereafter every 1-3 days, until fusion rate is filled to get P1 between umbilical cord after reaching 70-90% Matter stem cell；Then necessary passage is carried out according to above-mentioned cultural method；
(5) it freezes: addition cells frozen storing liquid in umbilical cord mesenchymal stem cells obtained by step (4) is freezed in liquid nitrogen, it is spare.
2. the method according to claim 1, the cells frozen storing liquid includes DMEM-F12, dimethyl sulfoxide and dog blood albumin； For example, the cells frozen storing liquid includes about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide, about 15 parts of dog blood albumin； For example, also adding dextran -40 in the frozen stock solution；For example, further including 0.2~0.5%w/v dextrose in the frozen stock solution Glycosides -40 preferably also includes 0.25%w/v dextran -40；For example, cells frozen storing liquid include about 65 parts DMEM-F12, about 10 Dimethyl sulfoxide, about 15 parts of the dog blood albumin, 0.2~0.5%w/v especially 0.25%w/v dextran -40 of part；Example Such as, the cells frozen storing liquid includes 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.03~0.05% W/v such as 0.04%w/v PLURONICS F87 and 0.5~0.7%w/v such as 0.6%w/v histidine.
3. the method that pair mescenchymal stem cell is frozen and recovered, method includes the following steps:
(51) mescenchymal stem cell being prepared is uniformly mixed with cells frozen storing liquid, then the cell liquid is placed in liquid nitrogen Freezing freezes the use until cell；
(52) when needing to make cell recovery, the cell frozen is removed from liquid nitrogen, is put into 37 DEG C of water-baths rapidly, 1~ Melt frozen stock solution completely in 5min, cell is transferred on 4 DEG C of ice baths after melting, and completes cell recovery.
4. according to the method in claim 3, wherein mescenchymal stem cell and cells frozen storing liquid are mixed with the ratio of volume ratio 1:1 's.
5. according to the method in claim 3, wherein the cells frozen storing liquid include 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.2~0.5%w/v especially 0.25%w/v dextran -40；For example, the cells frozen storing liquid packet Lip river is moored containing 65 parts of DMEM-F12,10 parts of dimethyl sulfoxides, 15 parts of dog blood albumin, 0.03~0.05%w/v such as 0.04%w/v Husky nurse 188 and 0.5~0.7%w/v such as 0.6%w/v histidine.
6. method according to claim 5, the cells frozen storing liquid is prepared according to such as under type: by the DMEM- of regulated proportion F12, dimethyl sulfoxide, dog blood albumin are uniformly mixed by volume, and the PLURONICS F87 and histidine of specified amount is added, makes Dissolution to get.
7. the cells frozen storing liquid of mesenchymal stem cell cryopreserving and recovery is used for, wherein including 65 parts of DMEM-F12,10 parts of dimethyl Sulfoxide, 15 parts of dog blood albumin, 0.03~0.05%w/v such as 0.04%w/v PLURONICS F87 and 0.5~0.7%w/v Such as 0.6%w/v histidine.
8. cells frozen storing liquid according to claim 7 is to prepare according to such as under type: by the DMEM-F12 of regulated proportion, two Methyl sulfoxide, dog blood albumin are uniformly mixed by volume, and the PLURONICS F87 and histidine of specified amount is added, makes to dissolve, i.e., ?.
9. cells frozen storing liquid according to claim 7, wherein the mescenchymal stem cell freeze and recover the following steps are included:
(51) (such as any one of digestion process, cell culture and cell succeeding generations, such as first aspect present invention institute will be passed through State step (1)~(4) in method) mescenchymal stem cell that is prepared is uniformly mixed with cells frozen storing liquid, and it is then that this is thin Cytosol is placed in liquid nitrogen and freezes, and freezes the use until cell；
(52) when needing to make cell recovery, the cell frozen is removed from liquid nitrogen, is put into 37 DEG C of water-baths rapidly, 1~ Melt frozen stock solution completely in 5min, cell is transferred on 4 DEG C of ice baths after melting, and completes cell recovery.
10. cells frozen storing liquid according to claim 7, wherein mescenchymal stem cell and cells frozen storing liquid are with volume ratio 1:1 Ratio mixing；Alternatively, any one of claim 1~10 mescenchymal stem cell is the fat or umbilical cord system for using dog or cat instead For what is obtained.
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