CN108795853A - Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell - Google Patents

Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell Download PDF

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CN108795853A
CN108795853A CN201810525167.8A CN201810525167A CN108795853A CN 108795853 A CN108795853 A CN 108795853A CN 201810525167 A CN201810525167 A CN 201810525167A CN 108795853 A CN108795853 A CN 108795853A
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stem cell
cell
mescenchymal stem
fetal membrane
days
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CN108795853B (en
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王小武
迟大明
朱春颖
郭春明
许晓椿
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Tianjin ruibosi Biotechnology Co.,Ltd.
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Weihai North Boya Species Diversity Research Institute Co Ltd
Tianjin Boya Xiu Yan Biotechnology Co Ltd
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N2500/32—Amino acids

Abstract

The present invention relates to the methods and dog fetal membrane mescenchymal stem cell that prepare dog fetal membrane mescenchymal stem cell.It is particularly used for the cells frozen storing liquid of dog fetal membrane mescenchymal stem cell, the method for preparing dog fetal membrane mescenchymal stem cell includes the following steps：Disinfection and cleaning, digestion process, cell culture, cell passage, cell cryopreservation.Excellent technique effect is presented in the method that the present invention prepares mescenchymal stem cell from dog fetal membrane, and culture medium used can greatly improve the efficiency for preparing stem cell.Obtained mescenchymal stem cell can be beneficial treatment canid 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.

Description

Prepare the method and dog fetal membrane mescenchymal stem cell of dog fetal membrane mescenchymal stem cell
Technical field
The invention belongs to the stem-cell therapy technical field of canid disease, it is related to preparing mesenchyma from the fetal membrane of dog The purposes of the method for stem cell and this mescenchymal stem cell in treating canid disease.The present invention is from dog fetal membrane system Excellent technique effect is presented in the method for standby mescenchymal stem cell.Obtained mescenchymal stem cell can be beneficial treatment Canidae It is the arthritis of animal, fracture, muscle damage, ligament injury, cartilage damage, joint injury, cognition dysfunction, immune mediating Disease, xerophthalmia, recurrent uveitis, liver diseases, heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid gland Disease, skin disease.Further, the invention further relates to frozen to the mescenchymal stem cell prepared from the fetal membrane of dog Method.The present invention additionally relates to the culture medium used during preparing dog fetal membrane mescenchymal stem cell, this culture medium Possibility is provided efficiently to obtain mescenchymal stem cell.The present invention is additionally related in a kind of jelly of dog fetal membrane mescenchymal stem cell Liquid storage.
Background technology
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, fetal membrane, marrow, adipose tissue.MSC have it is very strong from My proliferative capacity and multi-lineage potential.Under controlled condition, it can be divided into vivo or in vitro as nerve cell, cartilage are thin A plurality of types of cells such as born of the same parents, adipocyte, cardiac muscle cell and osteoblast.MSC also has immune compatibility, Bu Huizao simultaneously At immunological rejection, allograft can be carried out.In addition MSC does not have oncogenicity also, but can hematopoiesis support, and can secrete Multiple beneficial cell factor and immune factor, therefore regeneration and repairing and treating can be can be safely used for, in traditional medical means Huge treatment potential is shown in terms of helpless difficult and complicated cases.In veterinary applications, stem cell therapy can be carried effectively The quality of life of high animal helps them to break away from ailing puzzlement.Wherein dog is not only important companion animals, also can be trained As 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 experiment. Currently, in terms of dog stem-cell therapy, had it is many successful treatment dog arthritis is injected by mescenchymal stem cell, fracture, The case of muscle, ligament or cartilage/joint injury.Other such as canine cognition dysfunctions, immune-mediated disease, dry eyes Disease, recurrent uveitis, all kinds of liver diseases, heart disease, kidney trouble, diabetes, enterogastric diseases, thyroid gland problem, The treatment of skin 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 placenta/umbilical cord, fetal membrane belong to animal life Waste when production, but they are also abundant one of the sources MSC, therefore being used as sample separation cell by them can be more preferable Protect animal in ground.
At present from the organ-/ tissue of dog, especially from the fetal membrane of dog, the method that separation prepares mescenchymal stem cell is limited, And there is its limitation.Although detaching stem cell from mankind linked groups, organ has numerous method reports, such as the applicant The Chinese invention patent publication number CN102660501A (Chinese Patent Application No. 201210159918.1) of R&D team is disclosed A method of separation and amplification mesenchymal stem cell from fresh tissue of umbilical cord, however, it is possible to be due to species difference, originally Invention it was found by the inventors that the method for the above patent document is not appropriate for being directly used in canid.
Therefore, the method for having new method to prepare mescenchymal stem cell from the fetal membrane separation of dog is urgently expected in this field.
Invention content
The object of the present invention is to provide a kind of methods preparing mescenchymal stem cell from the fetal membrane of dog, unexpectedly It was found that preparing mescenchymal stem cell from the fetal membrane of dog using the method for the present invention, encouraging effect is presented, the present invention is based on this It was found that and being accomplished.
For this purpose, first aspect present invention provides method and/or the side of freezing for preparing mescenchymal stem cell from the fetal membrane of dog Method, this approach includes the following steps：
(1) it sterilizes and cleans：Placenta is taken out from Placenta samples collecting cassette, is placed in stainless steel cup, uses alcohol successively (such as 75% alcohol, alcohol soaking disinfection time are 10~60s, preferably 30s) and physiological saline rinsed surfaces repeatedly, to tire Disk carries out disinfection processing；Then it is slowly torn with surgical forceps and takes fetal membrane, be placed in glass dish, it is slow using phosphoric acid after rejecting blood vessel Fliud flushing (such as 0.025M phosphate sodium dihydrogen buffer solutions of pH6.5)) it rinses again, removal surface crimson blood, impurity；(this step is logical It is often to be handled in Biohazard Safety Equipment)
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block (in another cell culture plate) (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 I, DMEM-F12, for example, according to Following method is prepared：The collagenase type I of 0.1g is 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 of 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 Go out, adds appropriate (such as 3ml) mescenchymal stem cell culture medium, continue 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；It carries out once changing entirely per 1-3 days (such as 2 days) backward 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) by attached cell detachment vessel Supernatant is taken in bottom, centrifugation away, and mesenchymal stem cell media suspension cell again is added, is inoculated in culture vessel and is trained It supports, is hereafter changed the liquid once per 1-3 days (such as every 2 days), to get P1 generations after fusion rate reaches 70-90% (such as 80%) Fetal membrane mescenchymal stem cell；Then necessary passage is carried out (for example, used in the present invention according to above-mentioned cultural method TrypLETMExpress, consisting of：Potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, The rProtease of seven water disodium hydrogen phosphate 2160.0mg/L, EDTA 457.6mg/L, commercialization amount；The TrypLETMExpress Can be by commercial sources purchased from match Mo Feishier companies, relevant technical information is for example, see http:// www.thermofisher.com/cn/zh/home/technical-resources/media- formulation.346.html)；Optional
(5) it freezes：Will addition cells frozen storing liquid in fetal membrane mescenchymal stem cell 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 (DMSO) and people's blood Albumin.In one embodiment, the cells frozen storing liquid include about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide (DMSO), About 15 parts of human serum 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 A concentration of 0.01-0.5M of phosphate radical, preferably 0.02-0.1M in buffer solution.In following experiments of the present invention, do not say especially such as Bright, PBS buffer solution used is sodium ascorbyl phosphate, wherein a concentration of 0.025M of phosphate radical, pH 6.5.It should be noted that this hair A person of good sense has found that PBS buffer solution concentration and pH value within the above range is little for the influential effect of the method for the present invention.
According to method of the first aspect of the present invention, it is that collagenase type I is added enzyme solutions to be digested described in step (2) DMEM-F12 is obtained by filtration by filter, and digestive ferment 0.05g-0.5g, preferably digestive ferment are 0.08g-0.2g, excellent It is 0.1g to select digestive ferment, 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, mixing, and filtering (such as being filtered with 0.45um filters) 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 being highly preferred when about 0.1 cubic centimetre of 0.05-0.2 cubic centimetres, especially size.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 in experiments it is found that 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 more than after 1 cubic centimetre There is notable 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 within 1-2.5 hours digestion process time, disappear to tissue block It is best to change treatment effect, both can guarantee that tissue block obtains sufficient digestion process, was 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 carries 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), terminating the mescenchymal stem cell culture medium of digestion is According to 2:1~1:2 ratio is added, and preferably 1:1 ratio, the ratio are 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 centrifuge 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 FBS containing 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 containing 80-90% in mescenchymal stem cell culture medium.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 to 1 in Express digestive ferments, 2- propylene glycol.For example, the TrypLETMThe 1,2- propylene glycol of 0.05%w/v is also additionally added in Express digestive ferments.? Supplement propylene glycol in glycine and digestive ferment is augmented through having now surprisingly been found that, in mescenchymal stem cell culture medium to be helped to carry The yield of high 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 scheme, in the frozen stock solution, preferably also includes 0.25%w/v dextroses Glycosides -40.In one embodiment, which includes about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide (DMSO), about 15 parts of human serum albumin, 0.2~0.5%w/v especially 0.25%w/v dextrans -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, CO wherein in step (3) described incubator2A concentration of 3-7% is preferably dense Degree is 5%, and incubator temperature controls in body temperature environs, preferably 34-40 DEG C, preferably 36-38 DEG C, preferably 37 DEG C. Such CO2Concentration and temperature are usually also condition of culture commonly used in the art.
In addition, in the first aspect of the present invention, it is dry thin to provide separation and amplification mesenchyma from dog fetal membrane flesh tissue The method of born of the same parents.Therefore, second aspect of the present invention provides a kind of dog fetal membrane mescenchymal stem cell.
Dog fetal membrane mescenchymal stem cell according to a second aspect of the present invention is any implementation according to a first aspect of the present invention What scheme the method obtained.
Dog fetal membrane mescenchymal stem cell according to a second aspect of the present invention, cell purity are more than 85%, are greater than 90%.In one embodiment, the fetal membrane mescenchymal stem cell is after 3 more than generation pass on, and cell purity is more than 85%, example Such as larger than 90%.
Further, third aspect present invention is related to dog fetal membrane mescenchymal stem cell produced by the present invention in preparation for controlling Treat and/or prevent the purposes in the preparation selected from following canid disease：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 related to a kind of mescenchymal stem cell culture medium, wherein containing about 15 parts by weight FBS, the L-Glutamine of about 1 parts by weight, about 0.05 parts by weight Gentamicin and about 84 parts by weight DMEM-F12.
Mescenchymal stem cell culture medium according to a fourth aspect of the present invention, wherein also supplement has glycine.For example, between described The L-Glutamine of FBS, 1 parts by weight containing 15 parts by weight in mesenchymal stem cell media, 0.05 parts by weight DMEM-F12,0.025%w/v glycine of Gentamicin, 84 parts by weight.
Further, fifth aspect present invention is related to a kind of cells frozen storing liquid, wherein including about 65 parts of DMEM-F12, about 10 parts of dimethyl sulfoxide (DMSO), about 15 parts of human serum albumin, 0.2~0.5%w/v especially 0.25%w/v dextrans -40.
The present invention is further illustrated below.Cited text in document and the document cited in the present invention 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 of the either side of invention, 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 " fetal membrane mescenchymal stem cell " refers to the mescenchymal stem cell from fetal membrane.Therefore exist In the present invention, more particularly to the present invention context in, term " fetal membrane mescenchymal stem cell " can with " fetal membrane stem cell ", " 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 " fetal membrane " refers to the fetal membrane of dog, particularly relates to the fetal membrane 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, adipocyte, 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 Source channel genes expression.Therefore mescenchymal stem cell not still tissue-engineered bone, cartilage and cardiac muscle structure 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 It is separately cultured out mescenchymal stem cell in equal Various Tissues.
The method of the invention discloses a kind of from fetal membrane a large amount of separating mesenchymal stem cells, and protected using this method It deposits fetal membrane mescenchymal stem cell and establishes fetal membrane stem cell bank.The present inventor was separately cultured mesenchyma in summary and did in the past On the basis of cell, using tissue digestion enzymic digestion fetal membranes block, in conjunction with stationary culture, success is isolated from fetal membrane A large amount of mescenchymal stem cells.Mescenchymal stem cell purity that the method for the present invention obtains is high, quantity is more, has dry with medulla mesenchyma The identical biological characteristics of cell, can be to differentiation such as osteoblast, cartilage cell, adipocyte, endothelial cell, nerve cells. Since stem cell is clinically with a wide range of applications compared with adult stem cell naivety, rich content in fetal membrane, we use Conventional cell freezing method freezes mescenchymal stem cell as bleeding of the umbilicus, establishes fetal membrane stem cell bank, is later dry The further investigation of cell and clinical treatment lay the foundation.
Due to containing abundant candidate stem cell in bleeding of the umbilicus, people are this important biology of the fetal membrane candidate stem cell of dog Resource stores, and a kind for the treatment of means are provided for a variety of diseases in the blood system and disease of immune system.Same fetal membrane 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 preserved for a long time in the profound hypothermia liquid nitrogen of family name's degree, establishes fetal membrane stem cell bank, 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 fetal membrane 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 fetal membranes and side Method can succeed and effectively the full cell in tissue is separated.
The present invention is easy to operate, 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, adipocyte, cartilage cell, endothelial cell, nerve cell.Tire of the present invention success from dog Separation obtains a large amount of higher mescenchymal stem cells of purity in film, and can establish the fetal membrane stem cell bank 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 fetal membrane is as bleeding of the umbilicus, Cell Component is inmatureer, It derives from a wealth of sources, is conveniently easy to get, therefore the method for the present invention will above have extensive foreground in the canid application of stem cell.
Specific implementation mode
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 that are arrived used in experiment 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 fetal membrane of dog
(1) it sterilizes and cleans：Placenta is taken out from Placenta samples collecting cassette, is placed in stainless steel cup, uses alcohol successively (75% alcohol) and physiological saline rinsed surfaces (alcohol soaking disinfection time 30s) repeatedly), it carries out disinfection processing to placenta； Then slowly torn with surgical forceps and take fetal membrane, be placed in glass dish, reject blood vessel after, using phosphate buffer (pH6.5's 0.025M phosphate sodium dihydrogen buffer solutions)) it rinses again, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block in another cell culture plate Tissue block is put into digestion enzyme solutions (preparation method by (about 0.1 cubic centimetre of cube is blocky)：By the collagenase type I of 0.1g 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 (with volume ratio 1:1 ratio addition；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 centrifuges 10 minutes removal supernatants, is added PBS buffer solution is resuspended after cell again 1250rpm and centrifuges 10 minutes removal supernatants, and mescenchymal stem cell culture medium is added and (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 (CO2A concentration of 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；Per 1-3 days (this example, 2 days), progress once changed liquid entirely backward；
(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 fetal membrane mescenchymal stem cell；Then Necessary passage, which is carried out, according to above-mentioned cultural method (in this example, P15 generations is passaged to, wherein cell purity is more than after 3 generations passed on 95%；This example, commercial prod TrypLE usedTMExpress 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 companies, relevant technical information ginseng See http://www.thermofisher.com/cn/zh/home/technical-resources/media- formulation.346.html)；
(5) it freezes：Cells frozen storing liquid (1 will be added in fetal membrane mescenchymal stem cell obtained by step (4):1), cold in liquid nitrogen Freeze, it is spare (this example, cells frozen storing liquid used include 65 parts of DMEM-F12,10 parts of dimethyl sulfoxide (DMSO)s, 15 parts of human serum albumins).? Before freezing, by the details of mescenchymal stem cell (including dog age, kind of dog, hereditary information, immunization campaign information, viral diagnosis Information etc.) input computer data, it is for future reference to establish data archival.
Embodiment 2 prepares mescenchymal stem cell from the fetal membrane of dog
(1) it sterilizes and cleans：In Biohazard Safety Equipment, placenta is taken out from Placenta samples collecting cassette, is placed in stainless steel cup In, alcohol (75% alcohol) and physiological saline rinsed surfaces (alcohol soaking disinfection time 30s) repeatedly are used successively), to tire Disk carries out disinfection processing；Then it is slowly torn with surgical forceps and takes fetal membrane, be placed in glass dish, it is slow using phosphoric acid after rejecting blood vessel Fliud flushing (the 0.025M phosphate sodium dihydrogen buffer solutions of pH6.5)) it rinses again, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block in another cell culture plate Tissue block is put into digestion enzyme solutions (preparation method by (about 0.1 cubic centimetre of cube is blocky)：By the collagenase type I of 0.1g 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 (with volume ratio 1:1 ratio addition；In this example, 15 parts by weight are contained in mescenchymal stem cell culture medium DMEM-F12,0.025%w/v of FBS, the L-Glutamine of 1 parts by weight, the Gentamicin of 0.05 parts by weight, 84 parts by weight Glycine) to terminate digestion, then carrying out cell cleaning to the cell that digestion obtains, (in this example, 1250rpm is centrifuged 10 minutes and is gone Except supernatant, PBS buffer solution is added, 10 minutes removal supernatants of 1250rpm centrifugations 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 (CO2A concentration of 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；Per 1-3 days (this example, 2 days), progress once changed liquid entirely backward；
(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, and mesenchymal stem cell media suspension cell again is added, then It is inoculated in culture vessel to be cultivated, hereafter be changed the liquid once per 1-3 days (this example, every 2 days), until fusion rate reaches 70-90% To get P1 for fetal membrane mescenchymal stem cell after (this example up to 80%)；Then necessary passage (this example is carried out according to above-mentioned cultural method In, P15 generations are passaged to, wherein cell purity is more than 95% after 3 generations passed on；This example, TrypLE usedTMIts composition of Express For：Potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, seven water disodium hydrogen phosphates The rProtease of 2160.0mg/L, EDTA 457.6mg/L, commercialization amount；The TrypLETMExpress can pass through business way Diameter is purchased from match Mo Feishier companies, 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 (1 will be added in fetal membrane mescenchymal stem cell obtained by step (4):1), cold in liquid nitrogen Freeze, it is spare (this example, cells frozen storing liquid used include 65 parts of DMEM-F12,10 parts of dimethyl sulfoxide (DMSO)s, 15 parts of human serum albumins).? Before freezing, by the details of mescenchymal stem cell (including dog age, kind of dog, hereditary information, immunization campaign information, viral diagnosis Information etc.) input computer data, it is for future reference to establish data archival.
In the present embodiment 2, from step (1) to step (4), gained P1 is for dog fetal membrane mescenchymal stem cell, every gram of fetal membrane The average yield of tissue is 4~7 × 107In cell context (average result tested through 5 times, similarly hereinafter)；In the present embodiment 2, If do not augment glycine in mescenchymal stem cell culture medium, every gram fetal membranes of the P1 for dog fetal membrane mescenchymal stem cell Average yield is 6~8 × 105Cell context；In the present embodiment 2, if digestive ferment TrypLETM1 is not augmented in Express, 2- propylene glycol, then P1 for the average yield of every gram of fetal membranes of dog fetal membrane mescenchymal stem cell 5~7 × 105Cell context； This shows to augment glycine only in mescenchymal stem cell culture medium while in digestive ferment TrypLETM1 is augmented in Express, When 2- propylene glycol, the yield of dog fetal membrane mescenchymal stem cell can be just significantly improved.Similar, in embodiment 1, from step (1) it is tested to P1 obtained by step (4) through 5 times, the average yield of every gram of fetal membranes is 5 for dog fetal membrane mescenchymal stem cell ~8 × 105In cell context (when being handled people's fetal membranes in 1 same method of the embodiment of the present invention, every gram of fetal membrane group The average yield knitted can reach 7~8 × 107Cell context, big two orders of magnitude, this may be caused by due to species variation). The present inventor has found that the fetal membrane mesenchyma with reference to the method preparation dog of CN 102660501A embodiments 1 is dry thin in complementary testing When born of the same parents, P1 for every gram of fetal membranes of stem cell average yield 3~5 × 105In cell context；With reference to CN 106702499A In embodiment 1 to 5 method of embodiment when various scheme combinations, P1 is respectively less than 7 for the average yield of every gram of fetal membranes of stem cell ×105Cell.
Making dog fetal membrane mescenchymal stem cell obtained by the present invention, (formula is with conventional cryopreservation liquid：65 parts of DMEM-F12,10 part two Methyl sulfoxide, 15 parts of human serum albumins) freeze 30 days, then recovered with conventional method, measure freeze before cell number and Cell number after recovery, born of the same parents' number with cell number after recovery divided by before freezing is multiplied by using 100% gained percentage as survival hundred Score.Embodiment 1 gained P1 to P15 generations through freeze-percent survival of resuscitation process is in 63~75% ranges, such as implement The P3 of example 1 is 69% for the percent survival of dog fetal membrane mescenchymal stem cell；2 gained P1 of embodiment to P15 generations through freeze- The percent survival of resuscitation process in 65~73% ranges, such as embodiment 2 P5 for dog fetal membrane mescenchymal stem cell survival Percentage is 70%.
In the example supplemented at one, using improvement cells frozen storing liquid instead in 1 step of embodiment (5) (wherein includes：65 parts DMEM-F12,10 parts of dimethyl sulfoxide (DMSO)s, 15 parts of human serum albumins, 0.25%w/v dextrans -40), as a result show P1 to P15 For cell through freeze-percent survival of resuscitation process in 93~96% ranges, such as P3 is for dog fetal membrane mescenchymal stem cell Percent survival is 94%.In the example supplemented at one, improvement cells frozen storing liquid is used instead in 2 step of embodiment (5) (wherein Including：65 parts of DMEM-F12,10 parts of dimethyl sulfoxide (DMSO)s, 15 parts of human serum albumins, 0.25%w/v dextrans -40), as a result show Show P1 to P15 for cell through freeze-percent survival of resuscitation process in 92~96% ranges, such as P5 is for dog fetal membrane mesenchyma The percent survival of stem cell is 95%.It has now surprisingly been found that, can be shown when adding micro dextran in frozen stock solution Cell death caused by raising cell cryopreservation-resuscitation process of work.
The Identification of Biological Characteristics of embodiment 3, fetal membrane MSC
Following tests, the experiment of two kinds of stem cells are carried out to embodiment 1 and 2 gained dog fetal membrane mescenchymal stem cell 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 fetal membrane mescenchymal stem cell culture 48 hours Fusiformis attached cell can be obviously seen under micro mirror, can form within 8 days or so turbine-like cell clone, 75% can be formed 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 generations, form and growth characteristic are also without substantially changeing.
2, flow cytometry identification of M SC surface markers
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 measurement of the drafting of fetal membrane MSC growth curves and exponential phase doubling time
Logarithmic growth phase cell, digestion count, with the LG-DMEM culture mediums of 10%FBS be made cell suspension (2 × 104/ ml), it is inoculated with 0.5ml in 24 orifice plates per hole, 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 exponential phase, i.e. Td=Tlg2/Lg (Nt/ with Patterson formula No), Td：Doubling time (h), T：Cell increases to the time (h) used in Nt, N by No：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.According to formula calculate the 5th generation cell exponential phase of growth multiplication Time is within the scope of 24~33 hours.
4, the identification of fetal membrane MSC multi-lineage potentials
(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 mediums, use instead containing 10% through screening the DMEM-HG of FBS and 0.1 μM of dexamethasone, anti-bad being 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 tubercles are formed.Containing 0.1 μM of dexamethasone, 50 μM of ascorbyl phosphate, β-phosphoglycerol 5mM cultures are added in 10% DMEM-HG through screening FBS 1 week, apparent change occurred for cellular morphology, becomes polygonal from fusiform fibroblast sample, is similar to neuronal cell Sample, there is long filiform and protrudes 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 cultivating 4 weeks, it is seen that apparent calcium scoring.At 2 weeks Alkaline phosphatase staining is in strong positive reaction, reaches 94% 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 mediums, use instead containing 10% through screening the DMEM in high glucose of FBS, 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, coinduction 2 weeks, and oil red dyeing identification fat drips are formed.Containing 10% DMEM-HG through screening FBS, dexamethasone is added 0.5 μM, 50 μM of indocin, IBMX 0.5mM, 5 μ g/ml of insulin cultivate 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 gradually increase and merge, until when cultivating 2 weeks, it is seen that melt It closes pockets of fat drips and is full of entire cell.The fat generated in oil red O stain visible cell dyes red by specificity.
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, adipocytes, it was demonstrated that the dog mescenchymal stem cell tool that the method for the present invention obtains There are stem cell properties.
This method is easy to operate, stablizes, and the fetal membrane mescenchymal stem cell activity being thus prepared is high, can be used for dog routine The treatment of means refractory disease.

Claims (10)

1. for the cells frozen storing liquid of dog fetal membrane mescenchymal stem cell, wherein including about 65 parts of DMEM-F12, the two of about 10 parts Methyl sulfoxide, about 15 parts of human serum albumin, 0.2~0.5%w/v especially 0.25%w/v dextrans -40.
2. a kind of dog fetal membrane mescenchymal stem cell, cell purity is more than 90%；The dog fetal membrane mescenchymal stem cell is to shine to include What the method for following steps was prepared：
(1) it sterilizes and cleans：Placenta is used to alcohol and physiological saline rinsed surfaces repeatedly successively, is carried out disinfection place to placenta Reason；Then it tears and takes fetal membrane, after rejecting blood vessel, rinsed again using phosphate buffer, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block, 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 (for example, with density 0.2-2 × 104/cm2 It is added in culture vessel, preferably with density about 1 × 104/cm2It is added), then culture vessel is put into incubator and is cultivated, it trains Culture vessel is taken out from incubator when supporting to the 2-7 days (such as the 3-6 days, such as the 4th day, such as the 5th day), is added suitable (such as 3ml) mescenchymal stem cell culture medium is measured, continues to cultivate；At the 8-11 days (such as the 9th day) by culture vessel from training It supports and is taken out in case, carry out changing liquid entirely for the first time, continue to cultivate；It carries out once changing liquid entirely per 1-3 days (such as 2 days) backward；
(4) cell passes on：After the attached cell fusion rate in culture vessel reaches 40%-70% (such as 60%), utilize Digestive ferment is (for example, in the present invention, if not otherwise specified, use TrypLETMExpress) by attached cell detachment vessel bottom Supernatant is taken in portion, centrifugation away, and mesenchymal stem cell media suspension cell again is added, is inoculated in culture vessel and is cultivated, Hereafter it is changed the liquid once per 1-3 days (such as every 2 days), to get P1 for fetal membrane after fusion rate reaches 70-90% (such as 80%) Mescenchymal stem cell；Then necessary passage is carried out (for example, used in the present invention according to above-mentioned cultural method TrypLETMExpress, consisting of：Potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, The rProtease of seven water disodium hydrogen phosphate 2160.0mg/L, EDTA 457.6mg/L, commercialization amount；The TrypLETMExpress Can be by commercial sources purchased from match Mo Feishier companies, relevant technical information is for example, see http:// www.thermofisher.com/cn/zh/home/technical-resources/media- formulation.346.html)；Optional
(5) it freezes：Will addition cells frozen storing liquid in fetal membrane mescenchymal stem cell obtained by step (4) (such as with volume ratio 1:1 amount It is added) it is freezed in liquid nitrogen, it is spare.
3. preparing the method and/or cryopreservation methods of mescenchymal stem cell from the fetal membrane of dog, this approach includes the following steps：
(1) it sterilizes and cleans：Placenta is used to alcohol and physiological saline rinsed surfaces repeatedly successively, is carried out disinfection place to placenta Reason；Then it tears and takes fetal membrane, after rejecting blood vessel, rinsed again using phosphate buffer, removal surface crimson blood, impurity；
(2) digestion process：The fetal membranes that step (1) obtains are cut into tissue block, 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 (for example, with density 0.2-2 × 104/cm2 It is added in culture vessel, preferably with density about 1 × 104/cm2It is added), then culture vessel is put into incubator and is cultivated, it trains Culture vessel is taken out from incubator when supporting to the 2-7 days (such as the 3-6 days, such as the 4th day, such as the 5th day), is added suitable (such as 3ml) mescenchymal stem cell culture medium is measured, continues to cultivate；At the 8-11 days (such as the 9th day) by culture vessel from training It supports and is taken out in case, carry out changing liquid entirely for the first time, continue to cultivate；It carries out once changing liquid entirely per 1-3 days (such as 2 days) backward；
(4) cell passes on：After the attached cell fusion rate in culture vessel reaches 40%-70% (such as 60%), utilize Digestive ferment is (for example, in the present invention, if not otherwise specified, use TrypLETMExpress) by attached cell detachment vessel bottom Supernatant is taken in portion, centrifugation away, and mesenchymal stem cell media suspension cell again is added, is inoculated in culture vessel and is cultivated, Hereafter it is changed the liquid once per 1-3 days (such as every 2 days), to get P1 for fetal membrane after fusion rate reaches 70-90% (such as 80%) Mescenchymal stem cell；Then necessary passage is carried out (for example, used in the present invention according to above-mentioned cultural method TrypLETMExpress, consisting of：Potassium chloride 200.0mg/L, potassium dihydrogen phosphate 200.0mg/L, sodium chloride 8000.0mg/L, The rProtease of seven water disodium hydrogen phosphate 2160.0mg/L, EDTA 457.6mg/L, commercialization amount；The TrypLETMExpress Can be by commercial sources purchased from match Mo Feishier companies, relevant technical information is for example, see http:// www.thermofisher.com/cn/zh/home/technical-resources/media- formulation.346.html)；Optional
(5) it freezes：Will addition cells frozen storing liquid in fetal membrane mescenchymal stem cell obtained by step (4) (such as with volume ratio 1:1 amount It is added) it is freezed in liquid nitrogen, it is spare.
4. method according to claim 3, it is that DMEM-F12 is added in collagenase type I that enzyme solutions are digested described in step (2), is led to Cross what filter was obtained by filtration, digestive ferment 0.05g-0.5g, preferably digestive ferment are 0.08g-0.2g, and preferably digestive ferment is 0.1g, DMEM-F12 50-500ml, preferably DMEM-F12 are 80-200ml, and preferably DMEM-F12 is 100ml, filter For 0.45 μm of filter；For example, the digestion enzyme solutions are that the collagenase type I of 0.1g is added in the DMEM-F12 of 100ml, Mixing, filtering (such as being filtered with 0.45um filters) obtain.
5. in method according to claim 3, wherein step (2), the time of digestion process is 0.5-3 hours, and preferably 1-2.5 is small When, preferably 1.5-2 hours.
6. in method according to claim 3, wherein step (2), the mescenchymal stem cell culture medium for terminating digestion is according to 2:1 ~1:2 ratio is added, and preferably 1:1 ratio, the ratio are volume ratio.
7. method according to claim 3, the FBS containing about 15 parts by weight, about 1 weight in the mescenchymal stem cell culture medium The DMEM-F12 of the L-Glutamine of part, the Gentamicin and about 84 parts by weight of about 0.05 parts by weight；For example, wherein also increasing Benefit has glycine；For example, wherein also supplement has propylene glycol.
8. method according to claim 3, the cells frozen storing liquid includes about 65 parts of DMEM-F12, about 10 parts of dimethyl Asia Sulfone, about 15 parts of human serum albumin；For example, wherein also adding dextran；And/or wherein in step (3) described incubator CO2A concentration of 3-7%, preferred concentration 5%, incubator temperature control in body temperature environs, preferably 34-40 DEG C, It is preferred that 36-38 DEG C, preferably 37 DEG C.
9. a kind of mescenchymal stem cell culture medium, wherein the L-Glutamine of FBS, about 1 parts by weight containing about 15 parts by weight, The DMEM-F12 and optional glycine of the Gentamicin of about 0.05 parts by weight and about 84 parts by weight.
10. the dog fetal membrane that any one of the dog fetal membrane mescenchymal stem cell or claim 3-8 of claim 2 the method obtains Mescenchymal stem cell is being prepared for treating and/or preventing the purposes in the preparation selected from following canid disease：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.
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